http://2010.igem.org/wiki/index.php?title=Special:Contributions/Susanna&feed=atom&limit=50&target=Susanna&year=&month=2010.igem.org - User contributions [en]2024-03-29T05:55:41ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistryTeam:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry2010-10-28T03:31:40Z<p>Susanna: /* BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device */</p>
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<html><p align="center"><font size="4"><b>EXISTING PARTS FROM THE REGISTRY<br>(all these parts informations are already present in the registry) registr)</b></font></p></html><hr><br />
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[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization]<br />
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[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
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[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
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[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
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=Existing Parts from the Registry: list=<br />
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* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_R0010, BBa_R0011 - Wild type and hybrid lac promoters|BBa_R0010, BBa_R0011 - Wild type and hybrid lac promoters]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - R6K Origin of replication|BBa_J61001 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J2311 - constitutive promoters from Anderson's collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - chloramphenicol resistance cassette|BBa_P1004 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - GFP fusion brick|BBa_K125500 - GFP fusion brick]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
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<br><br />
----<br />
<br><br />
<br />
=<partinfo>BBa_R0010</partinfo>, <partinfo>BBa_R0011</partinfo> - Wild type and hybrid lac promoters=<br />
<partinfo>BBa_R0011</partinfo> hybrid lac promoter and the <partinfo>BBa_R0010</partinfo> wild type lac promoter were characterized at different copy number in TOP10 ''E. coli'' strain. This strain contains a lacI expression system in the genome.<br />
<br />
Induction static transfer function (computed in Relative Promoter Units), dynamics and metabolic burden were evaluated as a function of different IPTG concentrations in M9 supplemented with glycerol growth medium.<br />
<br />
A RFP generator (<partinfo>BBa_I13507</partinfo>) was used as a reporter gene. In particular, these measurement systems were used:<br />
<br />
*<partinfo>pSB1A2</partinfo>-<partinfo>BBa_J107010</partinfo><br />
*<partinfo>pSB1A3</partinfo>-<partinfo>BBa_J04450</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J107010</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J04450</partinfo><br />
<br />
At first, <partinfo>BBa_J107010</partinfo> and <partinfo>BBa_J04450</partinfo> inducibility was tested in a high copy number vector (<partinfo>pSB1A2</partinfo> or <partinfo>pSB1A3</partinfo>). The results are shown here as the relative RFP synthesis rate per cell.<br />
<br />
<br />
{|align="center"<br />
|[[Image:UNIPV_Pavia_r0010_1.png|500px|thumb|Relative RFP synthesis rate per cell in <partinfo>pSB1A2</partinfo>-<partinfo>BBa_J107010</partinfo> and <partinfo>pSB1A3</partinfo>-<partinfo>BBa_J04450</partinfo>. The error bars represent the standard errors of three independent measurements.]]<br />
|}<br />
<br />
<br />
Results show that in this condition <partinfo>BBa_R0010</partinfo> is about 2-fold stronger than <partinfo>BBa_R0011</partinfo>, but induced and uninduced cultures did not show differences in the RFP signal.<br />
<br />
This result is expected because the vectors are propagated at about 200 copies per cell, while the lacI repressor is present at single copy in the genome and thus it is not able to repress the lac promoters in such high copy.<br />
<br />
The doubling times and their standard errors estimated from data are reported below for <partinfo>pSB1A2</partinfo>-<partinfo>BBa_J107010</partinfo> and <partinfo>pSB1A3</partinfo>-<partinfo>BBa_J04450</partinfo> with and without 1mM of IPTG.<br />
<br />
<br />
{| width='80%' align='center' border='1'<br />
| '' Cultures'' || ''Mean doubling times [minutes]'' || ''standard errors over 3 independent experiment [minutes]''<br />
|-<br />
| <partinfo>pSB1A2</partinfo>-<partinfo>BBa_J107010</partinfo> || 77,7 || 3,1<br />
|-<br />
| <partinfo>pSB1A2</partinfo>-<partinfo>BBa_J107010</partinfo> + 1mM IPTG|| 76,5 || 2,2<br />
|-<br />
| <partinfo>pSB1A3</partinfo>-<partinfo>BBa_J04450</partinfo>|| 107,8 || 0,3<br />
|-<br />
| <partinfo>pSB1A3</partinfo>-<partinfo>BBa_J04450</partinfo> + 1mM IPTG|| 101,7 || 4,8<br />
|-<br />
|}<br />
<br />
<br />
These results demonstrate that cells growth is not significantly affected by the presence of IPTG, even at the high of 1 mM.<br />
<br />
<partinfo>BBa_J107010</partinfo> and <partinfo>BBa_J04450</partinfo> were then tested in the low copy (~5 copies per cell) vector <partinfo>pSB4C5</partinfo> in order to test their inducibility. The results are shown here as the RPU values at the steady state (constant RFP sysnthesis rate per cell) at different IPTG concentrations.<br />
<br />
{|align="center"<br />
|[[Image:UNIPV_Pavia_r0010_2.png|700px|thumb|RPU of <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J107010</partinfo> and <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J04450</partinfo> as a function of IPTG concentration. The error bars represent the standard errors of three independent measurements.]]<br />
|}<br />
<br />
Results show that in this condition both <partinfo>BBa_R0010</partinfo> and <partinfo>BBa_R0011</partinfo> produce different amounts of RFP as a function of the IPTG concentration. The amplitude of the two curves show that the promoters are very strong when induced with IPTG >= 10 uM. Although the experiments were carried out in the same conditions, the variability between experiments was high, especially for <partinfo>BBa_R0010</partinfo> (mean coefficient of variaton of about 37% for <partinfo>BBa_R0010</partinfo> and 15% for <partinfo>BBa_R0011</partinfo>), while the RPU variability between three wells in the same experiment is much lower (mean coefficient of variaton of bout 3.5% for both promoters).<br />
<br />
The above figure shows that <partinfo>BBa_R0011</partinfo> is stronger than the <partinfo>BBa_R0010</partinfo> wild type promoter in low copy plasmid. This result is unexpected because the same promoters in high copy vectors behaved differently (<partinfo>BBa_R0010</partinfo> was stronger than the <partinfo>BBa_R0011</partinfo>, see above).<br />
<br />
In the uninduced state, <partinfo>BBa_R0011</partinfo> has about the same strength as the <partinfo>BBa_J23101</partinfo> reference standard promoter.<br />
This static characteristic shows that the promoters are both leaky and a very low IPTG concentration (10 uM) is sufficient to trigger gene expression at *very* high levels.<br />
<br />
These results demonstrate that the genomic lacI is partially able to repress the two promoters, but very low IPTG concentrations are sufficient to bind the repressor and trigger the promoters transcription.<br />
<br />
Doubling times were also estimated for these cultures. Their values are reported below for uninduced and 1 mM IPTG-induced cultures.<br />
<br />
<br />
{| width='80%' align='center' border='1'<br />
| '' Cultures'' || ''Mean doubling times [minutes]'' || ''standard errors over 3 independent experiments [minutes]''<br />
|-<br />
| <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J107010</partinfo> || 113,5 || 10,8<br />
|-<br />
| <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J107010</partinfo> + 1mM IPTG|| 106,8 || 5,5<br />
|-<br />
| <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J04450</partinfo>|| 85 || 5<br />
|-<br />
| <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J04450</partinfo> + 1mM IPTG|| 90 || 4,5<br />
|-<br />
|}<br />
<br />
<br />
As obtained for the cultures with high copy plasmids, the growth rate of TOP10 harbouring low copy vectors with the measurement parts is not affected by IPTG presence.<br />
<br />
<br />
'''Dynamic characterization in low copy vector:''' The figure below shows a typical relative RFP synthesis rate per cell time series for <partinfo>BBa_J107010</partinfo> and <partinfo>BBa_J04450</partinfo> induced with 1 mM of IPTG and uninduced. These time series show that the full induction can be reached after about 50 min from the induction.<br />
<br />
<br />
{|align="center"<br />
|[[Image:UNIPV_Pavia_r0010_3.png|700px|thumb|Mean Scell signal as a function of time for <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J107010</partinfo> and <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J04450</partinfo>. Induced (with 1 mM of IPTG) and uninduced cultures are shown. Induction occurs at t=0. The shown graph is relative to one of the three experiments performed in different days.]]<br />
|}<br />
<br />
<br />
'''Conclusion:''' the characterization of two IPTG-inducible promoters has been performed and the performance of these two promoters have been compared in terms of transcriptional strength. The reported results are easily sharable in different laboratories thanks to the used standard RPU approach.<br />
<br />
<br />
'''Methods:'''<br />
*A of long term storage glycerol stock was streaked on a LB plate with suitable antibiotic. Tha plate was incubated overnight at 37°C.<br />
*A single colony was inoculated in 1 ml of M9 + suitable antibiotic in a 15 ml tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
*The grown cultures were then diluted 1:100 in 2-5 ml of M9 supplemented medium and incubated in the same conditions as before for about 4-5 hours.<br />
*For each desired IPTG concentration to be tested, three 200 ul aliquots of the cultures were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects in the frame).<br />
*2 ul of properly diluted IPTG (Sigma Aldrich) were added to the three wells for each desired concentration.<br />
*The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
**37°C constant for all the experiment;<br />
**sampling time of 5 minutes;<br />
**fluorescence gain of 50 or 80;<br />
**O.D. filter at 600 nm;<br />
**RFP filters at 535nm (ex) / 620nm (em);<br />
**15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
**Experiment duration time: about 6 hours.<br />
*This experiment was performed three times in different days.<br />
<br />
<br />
'''Data analysis:''' Relative Promoter Units (RPUs) were estimated as described by [Kelly JR et al. (2009), J Biol Eng 3:4].<br />
<br />
Briefly:<br />
*Absorbance and fluorescence time series were normalized by subtracting the absorbance of the media and the fluorescence of a negative control (a non fluorescent TOP10 culture) respectively, thus yielding O.D.600 and RFP time series.<br />
*RFP synthesis rate per cell (called ''Scell'') was computed as (1/O.D.600)*dGFP/dt. (this signal is not actually the RFP synthesis rate, but is proportional to it).<br />
*The RFP synthesis rate per cell was averaged at the steady state during the exponential growth phase (validated by identifying the linear region of the ln(O.D.600)).<br />
*The RPU of the promoter of interest in a specific condition was computed as ''mean_Scell,phi/mean_Scell,J23101'' where phi is the promoter of interest, J23101 is the reference standard and ''mean_Scell'' is the mean Scell signal value, computed as explained above.<br />
<br />
<br />
=<partinfo>BBa_K300009</partinfo>/<partinfo>BBa_I4102</partinfo> - PoPS->3OC6HSL sender device=<br />
<partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> was assembled downstream of the promoters the table reported in, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|} <br />
<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluorescent Protein) by <partinfo>BBa_F2620</partinfo> receiver device, to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced by autoinducer generators</b>===<br />
<br />
'''Experimental implementation:''' The autoinducer generators <partinfo>BBa_K300030</partinfo>, <partinfo>BBa_K300028</partinfo>, <partinfo>BBa_K300029</partinfo>, <partinfo>BBa_K300025</partinfo>, <partinfo>BBa_K300026</partinfo> and <partinfo>BBa_K300027</partinfo> were, thus, characterized by measuring the concentration of HSL released in the medium of cultures grown for 6 hours. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 biosensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results:''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table below:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced by autoinducer generators in high copy vector.]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> after a 6 hour cell growth is reported in figure and in the table below:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced by the autoinducer generators in low copy vector.]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
The signal generator was assembled on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized in tables.<br />
<br />
<br />
<div align='center>Sender/Receiver devices assembled as a unique BioBrick part on the same vector</div><br />
<br />
<div align='center>Sender devices assembled on low copy number vector and Receiver device on high copy number vector</div><br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of the receiver circuit. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center' width='80%'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing GFP were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]. An example of O.D.600 and fluorescence signals for a self-inducible device expressing GFP (<partinfo>BBa_K300026</partinfo>), as well as its Scell signal and the estimated threshold value, is reported below.<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|300px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|300px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|300px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|300px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest; it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]];<br />
*K_HSL is the HSL synthesis rate per cell; it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis to estimate the HSL synthesis rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double; it was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
'''Tab. 1 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br></td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300030.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
|Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br></td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300028.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300029</partinfo> (wiki name: I16) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300029.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.31 <br> *<br />
| 1.17 10^-16 <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300025.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300026.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300027.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 70%; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the algorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the O.D.start point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two modular PoPS-based devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were designed and used to realize a library of self-inducible devices, able to start the production of the heterologous protein at a defined culture density. They were characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A graphical summary is reported in the figures below:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Typical ''E. coli'' growth curve with O.D.start evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Typical ''E. coli'' growth curve with O.D.start evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell, and an algorithm was proposed in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In the figures below, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid).<br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the autoinducer production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by the reported graphs. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate is an increasing function of the upstream promoter's strength.<br />
<br />
For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably because too high luxI expression levels and/or synthesis rate of HSL are injurious for the cell.<br />
<br />
The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices able to perform autoinduction at many different O.D.600 values, in any cellular growth phase.<br />
<br />
=<partinfo>BBa_F2620</partinfo> - 3OC6HSL -> PoPS Receiver=<br />
=<partinfo>BBa_J61001</partinfo> - R6K Origin of replication=<br />
<br />
<partinfo>BBa_K300008</partinfo> was used as a validation construct for this conditional replication origin, in order to test its capability to be propagated in pir+ or pir-116 strain and its inability to propagate in the other ''E. coli'' strains.<br />
<br />
In particular, <partinfo>BBa_K300008</partinfo> was cut with XbaI-SpeI and the insert was isolated and purified from a 1% agarose gel. Then, it was self-ligated to generate a Cm-resistant R6K plasmid).<br />
<br />
BW25141 (<partinfo>BBa_K300984</partinfo>) and BW23474 (<partinfo>BBa_K300985</partinfo>) were chosen as pir+ and pir-116 strains respectively, while DH5alpha (<partinfo>BBa_V1001</partinfo>), MC1061 (<partinfo>BBa_K300078</partinfo>) and MG1655 (<partinfo>BBa_V1000</partinfo>) were chosen as pir- strains.<br />
<br />
<br />
All these strains were made competent following the commonly used CaCl2 method [Sambrook J, Fritsch EF, and Maniatis T (1989), Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.]. Then, a vial of 100 ul of competent cells was transformed with 2-4 ng of:<br />
*no DNA (negative control);<br />
*a pSB*** series vector (positive control);<br />
*self-ligated <partinfo>BBa_K300008</partinfo>.<br />
<br />
and plated on LB+Cm at 34 ug/ml for high-copy plasmids, Cm at 12.5 ug/ml for medium/low copy plasmids and for the negative control strains transformed with the R6K plasmids.<br />
<br />
<br />
The colonies were counted in each plate and the transformation efficiency was estimated in '''[CFU/ug of DNA]''' as:<br />
<br />
<div align=center><br />
''efficiency [CFU/ug of DNA]= # CFU * 1000 ng of DNA / amount of transformed DNA [ng]''<br />
</div><br />
<br />
The results are shown here:<br />
{|border=1<br />
|'''Strain'''<br />
|'''Efficiency with no DNA'''<br />
|'''Efficiency with pSB*** (positive control)'''<br />
|'''Efficiency with the self-ligated <partinfo>BBa_K300008</partinfo> (R6K plasmid)'''<br />
|-<br />
|<partinfo>BBa_K300084</partinfo><br />
|0<br />
|10^5<br />
|10^5<br />
|-<br />
|<partinfo>BBa_K300085</partinfo><br />
|0<br />
|10^6<br />
|10^6<br />
|-<br />
|<partinfo>BBa_V1001</partinfo><br />
|0<br />
|10^8<br />
|0<br />
|-<br />
|<partinfo>BBa_K300078</partinfo><br />
|0<br />
|10^6<br />
|0<br />
|-<br />
|<partinfo>BBa_V1000</partinfo><br />
|0<br />
|10^5<br />
|0<br />
|}<br />
<br />
These results show that <partinfo>BBa_J61001</partinfo> replication origin can be only propagated in pir+ and pir-116 strains (<partinfo>BBa_K300084</partinfo> and <partinfo>BBa_K300085</partinfo>), while the transformation of other strains with the R6K plasmid yielded no colonies after transformation.<br />
<br />
Moreover, these results show that the R6K plasmid in pir+ and pir-116 strains was transformed with the same efficiency as the pSB*** positive control plasmid, demonstrating that the R6K origin doesn't give any handicap in plasmid transformation.<br />
<br />
=<partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> - constitutive promoters from Anderson's collection=<br />
<br />
The <partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> were charcterized in LB and M9 supplemented with glycerol (0.4%) growth media in high copy and low copy vectors in ''E. coli'' TOP10 (<partinfo>BBa_V1009</partinfo>).<br />
<br />
RPU and doubling time were characterized for all of them, according to the protocols reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|this section]]. <br />
<br />
The following measurement systems were used for high copy plasmids:<br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23100</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23101</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23105</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23106</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23110</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23114</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23116</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23118</partinfo><br />
<br />
In order to build low copy plasmid measurement systems, the EcoRI-PstI fragment (J231xx-RFP) of each <partinfo>BBa_J61002</partinfo>-BBa_J231xx was assembled into <partinfo>pSB4C5</partinfo> vector. This fragment contains the constitutive promoter of interest upstream a RBS-RFP-TT expression system.<br />
<br />
The following measurement parts were used for low copy plasmids:<br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23100</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23101</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23105</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23106</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23110</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23114</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23116</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23118</partinfo><br />
<br />
<br />
The RPU values and doubling times are here reported:<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI fragment of <partinfo>BBa_J61002</partinfo>-BBa_J231xx in <partinfo>pSB4C5</partinfo> vector, in order to transfer the promoter and the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
The error bars represent the standard deviation for three dfferent wells in the same experiment.<br />
Doubling times were evaluated for the described cultures (HC stands for High Copy and LC stands for Low Copy):<br />
{| align='center' border='1'<br />
|rowspan='2'|<b>Promoter</b><br />
|colspan='3'| <b>doubling time [minutes]</b><br />
|-<br />
| LB in HC plasmid || M9 in HC plasmid || M9 in LC plasmid<br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 33.75 <br> ± <br> 1.34 || 82.53 <br> ± <br> 2.45 || 86.11 <br> ± <br> 4.45<br />
|-<br />
|<partinfo>BBa_J23101</partinfo> || 35.93 <br> ± <br> 0.62 || 82.68 <br> ± <br> 1.84 || 86.42 <br> ± <br> 1.91<br />
|-<br />
|<partinfo>BBa_J23105</partinfo> || 29.86 <br> ± <br> 0.33 || 63.09 <br> ± <br> 7.08 || 85.00 <br> ± <br> 5.13<br />
|-<br />
|<partinfo>BBa_J23106</partinfo> || 29.17 <br> ± <br> 0.96 || 68.11 <br> ± <br> 4.25 || 88.71 <br> ± <br> 0.90<br />
|-<br />
|<partinfo>BBa_J23110</partinfo> || 31.28 <br> ± <br> 0.42 || 67.52 <br> ± <br> 5.87 || 76.15 <br> ± <br> 2.16<br />
|-<br />
|<partinfo>BBa_J23114</partinfo> || 28.97 <br> ± <br> 0.49 || 59.44 <br> ± <br> 5.20 || 80.12 <br> ± <br> 0.95<br />
|-<br />
|<partinfo>BBa_J23116</partinfo> || 28.14 <br> ± <br> 0.25 || 72.74 <br> ± <br> 0.37 || 81.68 <br> ± <br> 3.08<br />
|-<br />
|<partinfo>BBa_J23118</partinfo> || 32.84 <br> ± <br> 0.31 || 73.64 <br> ± <br> 2.41 || 89.86 <br> ± <br> 2.93<br />
|}<br />
<br />
It was not possible to evaluate promoters activities in low copy number plasmids in LB because the RFP activity was too weak and not distinguishable from the background.<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the tested conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
<br />
<br />
=<partinfo>BBa_P1004</partinfo> - chloramphenicol resistance cassette=<br />
<br />
<partinfo>BBa_P1004</partinfo> has been successfully used in the assembly of <partinfo>BBa_K300000</partinfo> integrative base vector for ''E. coli''. All the intermediate parts which contained <partinfo>BBa_P1004</partinfo> showed Chloramphenicol resistance (tested up to 34 ug/ml in LB media) when transformed in TOP10 (<partinfo>BBa_V1009</partinfo>), DH5alpha (<partinfo>BBa_V1001</partinfo>), MC1061 (<partinfo>BBa_K300078</partinfo>), MG1655 (<partinfo>BBa_V1000</partinfo>), BW23474 (<partinfo>BBa_K300985</partinfo>) and DB3.1 (<partinfo>BBa_V1005</partinfo>) strains in a high-copy plasmid.<br />
<br />
<br />
=<partinfo>BBa_K125500</partinfo> - GFP fusion brick=<br />
This part can be useful to construct fluorescent fusion proteins. It is composed by a tail domain (the GFP <partinfo>K125500</partinfo>) with a transcriptional terminator (<partinfo>BBa_B0015</partinfo>) downstream. <br />
<br />
Other protein domains can be fused upstream of this part in order to create chimeric fluorescent proteins, or it can be ligated to tags useful for low-cost protein purification.<br />
<br />
This part was used do design the following BioBrick measurement systems:<br />
<br />
*<partinfo>BBa_K300086</partinfo><br />
*<partinfo>BBa_K300088</partinfo><br />
*<partinfo>BBa_K300090</partinfo><br />
*<partinfo>BBa_K300091</partinfo><br />
*<partinfo>BBa_K300092</partinfo><br />
*<partinfo>BBa_K300099</partinfo><br />
<br />
to test these contructs:<br />
<br />
*<partinfo>BBa_K300002</partinfo><br />
*<partinfo>BBa_K300093</partinfo><br />
*<partinfo>BBa_K300094</partinfo><br />
*<partinfo>BBa_K300095</partinfo><br />
*<partinfo>BBa_K300084</partinfo><br />
*<partinfo>BBa_K300097</partinfo><br />
<br />
respectively. All of the tested parts are synthetic fusion tags whose activity could be measured by assembling a promoter with RBS upstream and a tail domain with terminator downstream, thus yielding the measurement systems. <partinfo>BBa_K300005</partinfo> was used as a tail domain with terminator downstream. It has been assembled to test the correct folding of the resulting fusion protein by measuring the GFP and to test the affinity tag performance with a proof of concept protein.<br />
<br />
<br />
In all of the measurement parts, GFP could be successfully detected in bacteria harbouring the measurement parts in high copy plasmids. In this condition, GFP was detected by using an excitation filter at 485nm and an emission filter at 540nm in a Infinite F200 microplate reader (Tecan).<br />
<br />
<table><br />
<tr><br />
<td>[[Image:UNIPV10_pTET_c_GFP.png|thumb|300px|Raw GFP curve]]</td><br />
<td>[[Image:UNIPV10_pTET_c_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]</td><br />
</tr><br />
<tr><br />
<td>[[Image:UNIPV10_HSL_c_GFP.png|thumb|300px|Raw GFP curve]]</td><br />
<td>[[Image:UNIPV10_HSL_c_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]</td><br />
</tr><br />
</table><br />
<br />
=<partinfo>BBa_J72008</partinfo> - phi80 integration helper plasmid pInt80-649=<br />
<br />
<partinfo>BBa_J72008</partinfo> has been successfully used in the integration protocol of both MC1061 (<partinfo>BBa_K300078</partinfo>) and MG1655 (<partinfo>BBa_V1000</partinfo>) ''E. coli'' strains. See [http://partsregistry.org/Part:BBa_K300000:Experience BBa_K300000 Experience page] for details about how this plasmid was used.<br />
<br />
It can be actually cured at 37-42°C, while it can be propagated at 30°C.<br />
<br />
It actually enables the propagation of R6K (conditional replication origin) plasmids, thanks to its pir-116 gene.<br />
<br />
<tr><td><br></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistryTeam:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry2010-10-28T00:40:31Z<p>Susanna: </p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><td valign=top width="70%"><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<html><p align="center"><font size="4"><b>EXISTING PARTS FROM THE REGISTRY<br>(all these parts informations are already present in the registry) registr)</b></font></p></html><hr><br />
<br />
<tr><td width="100%"><br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><br />
<td align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization]<br />
</td><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th><br />
</tr><br />
</table><br><br />
<br />
=Existing Parts from the Registry: list=<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_R0010, BBa_R0011 - Wild type and hybrid lac promoters|BBa_R0010, BBa_R0011 - Wild type and hybrid lac promoters]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - R6K Origin of replication|BBa_J61001 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J2311 - constitutive promoters from Anderson's collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - chloramphenicol resistance cassette|BBa_P1004 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - GFP fusion brick|BBa_K125500 - GFP fusion brick]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=<partinfo>BBa_R0010</partinfo>, <partinfo>BBa_R0011</partinfo> - Wild type and hybrid lac promoters=<br />
<partinfo>BBa_R0011</partinfo> hybrid lac promoter and the <partinfo>BBa_R0010</partinfo> wild type lac promoter were characterized at different copy number in TOP10 ''E. coli'' strain. This strain contains a lacI expression system in the genome.<br />
<br />
Induction static transfer function (computed in Relative Promoter Units), dynamics and metabolic burden were evaluated as a function of different IPTG concentrations in M9 supplemented with glycerol growth medium.<br />
<br />
A RFP generator (<partinfo>BBa_I13507</partinfo>) was used as a reporter gene. In particular, these measurement systems were used:<br />
<br />
*<partinfo>pSB1A2</partinfo>-<partinfo>BBa_J107010</partinfo><br />
*<partinfo>pSB1A3</partinfo>-<partinfo>BBa_J04450</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J107010</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J04450</partinfo><br />
<br />
At first, <partinfo>BBa_J107010</partinfo> and <partinfo>BBa_J04450</partinfo> inducibility was tested in a high copy number vector (<partinfo>pSB1A2</partinfo> or <partinfo>pSB1A3</partinfo>). The results are shown here as the relative RFP synthesis rate per cell.<br />
<br />
<br />
{|align="center"<br />
|[[Image:UNIPV_Pavia_r0010_1.png|500px|thumb|Relative RFP synthesis rate per cell in <partinfo>pSB1A2</partinfo>-<partinfo>BBa_J107010</partinfo> and <partinfo>pSB1A3</partinfo>-<partinfo>BBa_J04450</partinfo>. The error bars represent the standard errors of three independent measurements.]]<br />
|}<br />
<br />
<br />
Results show that in this condition <partinfo>BBa_R0010</partinfo> is about 2-fold stronger than <partinfo>BBa_R0011</partinfo>, but induced and uninduced cultures did not show differences in the RFP signal.<br />
<br />
This result is expected because the vectors are propagated at about 200 copies per cell, while the lacI repressor is present at single copy in the genome and thus it is not able to repress the lac promoters in such high copy.<br />
<br />
The doubling times and their standard errors estimated from data are reported below for <partinfo>pSB1A2</partinfo>-<partinfo>BBa_J107010</partinfo> and <partinfo>pSB1A3</partinfo>-<partinfo>BBa_J04450</partinfo> with and without 1mM of IPTG.<br />
<br />
<br />
{| width='80%' align='center' border='1'<br />
| '' Cultures'' || ''Mean doubling times [minutes]'' || ''standard errors over 3 independent experiment [minutes]''<br />
|-<br />
| <partinfo>pSB1A2</partinfo>-<partinfo>BBa_J107010</partinfo> || 77,7 || 3,1<br />
|-<br />
| <partinfo>pSB1A2</partinfo>-<partinfo>BBa_J107010</partinfo> + 1mM IPTG|| 76,5 || 2,2<br />
|-<br />
| <partinfo>pSB1A3</partinfo>-<partinfo>BBa_J04450</partinfo>|| 107,8 || 0,3<br />
|-<br />
| <partinfo>pSB1A3</partinfo>-<partinfo>BBa_J04450</partinfo> + 1mM IPTG|| 101,7 || 4,8<br />
|-<br />
|}<br />
<br />
<br />
These results demonstrate that cells growth is not significantly affected by the presence of IPTG, even at the high of 1 mM.<br />
<br />
<partinfo>BBa_J107010</partinfo> and <partinfo>BBa_J04450</partinfo> were then tested in the low copy (~5 copies per cell) vector <partinfo>pSB4C5</partinfo> in order to test their inducibility. The results are shown here as the RPU values at the steady state (constant RFP sysnthesis rate per cell) at different IPTG concentrations.<br />
<br />
{|align="center"<br />
|[[Image:UNIPV_Pavia_r0010_2.png|700px|thumb|RPU of <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J107010</partinfo> and <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J04450</partinfo> as a function of IPTG concentration. The error bars represent the standard errors of three independent measurements.]]<br />
|}<br />
<br />
Results show that in this condition both <partinfo>BBa_R0010</partinfo> and <partinfo>BBa_R0011</partinfo> produce different amounts of RFP as a function of the IPTG concentration. The amplitude of the two curves show that the promoters are very strong when induced with IPTG >= 10 uM. Although the experiments were carried out in the same conditions, the variability between experiments was high, especially for <partinfo>BBa_R0010</partinfo> (mean coefficient of variaton of about 37% for <partinfo>BBa_R0010</partinfo> and 15% for <partinfo>BBa_R0011</partinfo>), while the RPU variability between three wells in the same experiment is much lower (mean coefficient of variaton of bout 3.5% for both promoters).<br />
<br />
The above figure shows that <partinfo>BBa_R0011</partinfo> is stronger than the <partinfo>BBa_R0010</partinfo> wild type promoter in low copy plasmid. This result is unexpected because the same promoters in high copy vectors behaved differently (<partinfo>BBa_R0010</partinfo> was stronger than the <partinfo>BBa_R0011</partinfo>, see above).<br />
<br />
In the uninduced state, <partinfo>BBa_R0011</partinfo> has about the same strength as the <partinfo>BBa_J23101</partinfo> reference standard promoter.<br />
This static characteristic shows that the promoters are both leaky and a very low IPTG concentration (10 uM) is sufficient to trigger gene expression at *very* high levels.<br />
<br />
These results demonstrate that the genomic lacI is partially able to repress the two promoters, but very low IPTG concentrations are sufficient to bind the repressor and trigger the promoters transcription.<br />
<br />
Doubling times were also estimated for these cultures. Their values are reported below for uninduced and 1 mM IPTG-induced cultures.<br />
<br />
<br />
{| width='80%' align='center' border='1'<br />
| '' Cultures'' || ''Mean doubling times [minutes]'' || ''standard errors over 3 independent experiments [minutes]''<br />
|-<br />
| <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J107010</partinfo> || 113,5 || 10,8<br />
|-<br />
| <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J107010</partinfo> + 1mM IPTG|| 106,8 || 5,5<br />
|-<br />
| <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J04450</partinfo>|| 85 || 5<br />
|-<br />
| <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J04450</partinfo> + 1mM IPTG|| 90 || 4,5<br />
|-<br />
|}<br />
<br />
<br />
As obtained for the cultures with high copy plasmids, the growth rate of TOP10 harbouring low copy vectors with the measurement parts is not affected by IPTG presence.<br />
<br />
<br />
'''Dynamic characterization in low copy vector:''' The figure below shows a typical relative RFP synthesis rate per cell time series for <partinfo>BBa_J107010</partinfo> and <partinfo>BBa_J04450</partinfo> induced with 1 mM of IPTG and uninduced. These time series show that the full induction can be reached after about 50 min from the induction.<br />
<br />
<br />
{|align="center"<br />
|[[Image:UNIPV_Pavia_r0010_3.png|700px|thumb|Mean Scell signal as a function of time for <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J107010</partinfo> and <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J04450</partinfo>. Induced (with 1 mM of IPTG) and uninduced cultures are shown. Induction occurs at t=0. The shown graph is relative to one of the three experiments performed in different days.]]<br />
|}<br />
<br />
<br />
'''Conclusion:''' the characterization of two IPTG-inducible promoters has been performed and the performance of these two promoters have been compared in terms of transcriptional strength. The reported results are easily sharable in different laboratories thanks to the used standard RPU approach.<br />
<br />
<br />
'''Methods:'''<br />
*A of long term storage glycerol stock was streaked on a LB plate with suitable antibiotic. Tha plate was incubated overnight at 37°C.<br />
*A single colony was inoculated in 1 ml of M9 + suitable antibiotic in a 15 ml tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
*The grown cultures were then diluted 1:100 in 2-5 ml of M9 supplemented medium and incubated in the same conditions as before for about 4-5 hours.<br />
*For each desired IPTG concentration to be tested, three 200 ul aliquots of the cultures were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects in the frame).<br />
*2 ul of properly diluted IPTG (Sigma Aldrich) were added to the three wells for each desired concentration.<br />
*The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
**37°C constant for all the experiment;<br />
**sampling time of 5 minutes;<br />
**fluorescence gain of 50 or 80;<br />
**O.D. filter at 600 nm;<br />
**RFP filters at 535nm (ex) / 620nm (em);<br />
**15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
**Experiment duration time: about 6 hours.<br />
*This experiment was performed three times in different days.<br />
<br />
<br />
'''Data analysis:''' Relative Promoter Units (RPUs) were estimated as described by [Kelly JR et al. (2009), J Biol Eng 3:4].<br />
<br />
Briefly:<br />
*Absorbance and fluorescence time series were normalized by subtracting the absorbance of the media and the fluorescence of a negative control (a non fluorescent TOP10 culture) respectively, thus yielding O.D.600 and RFP time series.<br />
*RFP synthesis rate per cell (called ''Scell'') was computed as (1/O.D.600)*dGFP/dt. (this signal is not actually the RFP synthesis rate, but is proportional to it).<br />
*The RFP synthesis rate per cell was averaged at the steady state during the exponential growth phase (validated by identifying the linear region of the ln(O.D.600)).<br />
*The RPU of the promoter of interest in a specific condition was computed as ''mean_Scell,phi/mean_Scell,J23101'' where phi is the promoter of interest, J23101 is the reference standard and ''mean_Scell'' is the mean Scell signal value, computed as explained above.<br />
<br />
<br />
=<partinfo>BBa_K300009</partinfo>/<partinfo>BBa_I4102</partinfo> - PoPS->3OC6HSL sender device=<br />
<partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> was assembled downstream of the promoters the table reported in, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these device because they produced amounts of LuxI protein that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformants could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluorescent Protein) by <partinfo>BBa_F2620</partinfo> receiver device, to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced by autoinducer generators</b>===<br />
<br />
'''Experimental implementation:''' The autoinducer generators <partinfo>BBa_K300030</partinfo>, <partinfo>BBa_K300028</partinfo>, <partinfo>BBa_K300029</partinfo>, <partinfo>BBa_K300025</partinfo>, <partinfo>BBa_K300026</partinfo> and <partinfo>BBa_K300027</partinfo> were, thus, characterized by measuring the concentration of HSL released in the medium of cultures grown for 6 hours. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 biosensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results:''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table below:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced by autoinducer generators in high copy vector.]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> after a 6 hour cell growth is reported in Fig.9 and in the table below:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced by the autoinducer generators in low copy vector.]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
The signal generator was assembled on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized in tables.<br />
<br />
<br />
<div align='center>Sender/Receiver devices assembled as a unique BioBrick part on the same vector</div><br />
<br />
<div align='center>Sender devices assembled on low copy number vector and Receiver device on high copy number vector</div><br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of the receiver circuit. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center' width='80%'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing GFP were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]. An example of O.D.600 and fluorescence signals for a self-inducible device expressing GFP (<partinfo>BBa_K300026</partinfo>), as well as its Scell signal and the estimated threshold value, is reported below.<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|300px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|300px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|300px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|300px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest; it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]];<br />
*K_HSL is the HSL synthesis rate per cell; it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis to estimate the HSL synthesis rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double; it was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
'''Tab. 1 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br></td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300030.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
|Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br></td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300028.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300029</partinfo> (wiki name: I16) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300029.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.31 <br> *<br />
| 1.17 10^-16 <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300025.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300026.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300027.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 70%; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the algorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the O.D.start point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two modular PoPS-based devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were designed and used to realize a library of self-inducible devices, able to start the production of the heterologous protein at a defined culture density. They were characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A graphical summary is reported in the figures below:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Typical ''E. coli'' growth curve with O.D.start evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Typical ''E. coli'' growth curve with O.D.start evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell, and an algorithm was proposed in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In the figures below, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid).<br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the autoinducer production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by the reported graphs. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate is an increasing function of the upstream promoter's strength.<br />
<br />
For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably because too high luxI expression levels and/or synthesis rate of HSL are injurious for the cell.<br />
<br />
The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices able to perform autoinduction at many different O.D.600 values, in any cellular growth phase.<br />
=<partinfo>BBa_F2620</partinfo> - 3OC6HSL -> PoPS Receiver=<br />
=<partinfo>BBa_J61001</partinfo> - R6K Origin of replication=<br />
<br />
<partinfo>BBa_K300008</partinfo> was used as a validation construct for this conditional replication origin, in order to test its capability to be propagated in pir+ or pir-116 strain and its inability to propagate in the other ''E. coli'' strains.<br />
<br />
In particular, <partinfo>BBa_K300008</partinfo> was cut with XbaI-SpeI and the insert was isolated and purified from a 1% agarose gel. Then, it was self-ligated to generate a Cm-resistant R6K plasmid).<br />
<br />
BW25141 (<partinfo>BBa_K300984</partinfo>) and BW23474 (<partinfo>BBa_K300985</partinfo>) were chosen as pir+ and pir-116 strains respectively, while DH5alpha (<partinfo>BBa_V1001</partinfo>), MC1061 (<partinfo>BBa_K300078</partinfo>) and MG1655 (<partinfo>BBa_V1000</partinfo>) were chosen as pir- strains.<br />
<br />
<br />
All these strains were made competent following the commonly used CaCl2 method [Sambrook J, Fritsch EF, and Maniatis T (1989), Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.]. Then, a vial of 100 ul of competent cells was transformed with 2-4 ng of:<br />
*no DNA (negative control);<br />
*a pSB*** series vector (positive control);<br />
*self-ligated <partinfo>BBa_K300008</partinfo>.<br />
<br />
and plated on LB+Cm at 34 ug/ml for high-copy plasmids, Cm at 12.5 ug/ml for medium/low copy plasmids and for the negative control strains transformed with the R6K plasmids.<br />
<br />
<br />
The colonies were counted in each plate and the transformation efficiency was estimated in '''[CFU/ug of DNA]''' as:<br />
<br />
<div align=center><br />
''efficiency [CFU/ug of DNA]= # CFU * 1000 ng of DNA / amount of transformed DNA [ng]''<br />
</div><br />
<br />
The results are shown here:<br />
{|border=1<br />
|'''Strain'''<br />
|'''Efficiency with no DNA'''<br />
|'''Efficiency with pSB*** (positive control)'''<br />
|'''Efficiency with the self-ligated <partinfo>BBa_K300008</partinfo> (R6K plasmid)'''<br />
|-<br />
|<partinfo>BBa_K300084</partinfo><br />
|0<br />
|10^5<br />
|10^5<br />
|-<br />
|<partinfo>BBa_K300085</partinfo><br />
|0<br />
|10^6<br />
|10^6<br />
|-<br />
|<partinfo>BBa_V1001</partinfo><br />
|0<br />
|10^8<br />
|0<br />
|-<br />
|<partinfo>BBa_K300078</partinfo><br />
|0<br />
|10^6<br />
|0<br />
|-<br />
|<partinfo>BBa_V1000</partinfo><br />
|0<br />
|10^5<br />
|0<br />
|}<br />
<br />
These results show that <partinfo>BBa_J61001</partinfo> replication origin can be only propagated in pir+ and pir-116 strains (<partinfo>BBa_K300084</partinfo> and <partinfo>BBa_K300085</partinfo>), while the transformation of other strains with the R6K plasmid yielded no colonies after transformation.<br />
<br />
Moreover, these results show that the R6K plasmid in pir+ and pir-116 strains was transformed with the same efficiency as the pSB*** positive control plasmid, demonstrating that the R6K origin doesn't give any handicap in plasmid transformation.<br />
<br />
=<partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> - constitutive promoters from Anderson's collection=<br />
<br />
The <partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> were charcterized in LB and M9 supplemented with glycerol (0.4%) growth media in high copy and low copy vectors in ''E. coli'' TOP10 (<partinfo>BBa_V1009</partinfo>).<br />
<br />
RPU and doubling time were characterized for all of them, according to the protocols reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|this section]]. <br />
<br />
The following measurement systems were used for high copy plasmids:<br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23100</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23101</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23105</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23106</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23110</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23114</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23116</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23118</partinfo><br />
<br />
In order to build low copy plasmid measurement systems, the EcoRI-PstI fragment (J231xx-RFP) of each <partinfo>BBa_J61002</partinfo>-BBa_J231xx was assembled into <partinfo>pSB4C5</partinfo> vector. This fragment contains the constitutive promoter of interest upstream a RBS-RFP-TT expression system.<br />
<br />
The following measurement parts were used for low copy plasmids:<br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23100</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23101</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23105</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23106</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23110</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23114</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23116</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23118</partinfo><br />
<br />
<br />
The RPU values and doubling times are here reported:<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI fragment of <partinfo>BBa_J61002</partinfo>-BBa_J231xx in <partinfo>pSB4C5</partinfo> vector, in order to transfer the promoter and the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
The error bars represent the standard deviation for three dfferent wells in the same experiment.<br />
Doubling times were evaluated for the described cultures (HC stands for High Copy and LC stands for Low Copy):<br />
{| align='center' border='1'<br />
|rowspan='2'|<b>Promoter</b><br />
|colspan='3'| <b>doubling time [minutes]</b><br />
|-<br />
| LB in HC plasmid || M9 in HC plasmid || M9 in LC plasmid<br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 33.75 <br> ± <br> 1.34 || 82.53 <br> ± <br> 2.45 || 86.11 <br> ± <br> 4.45<br />
|-<br />
|<partinfo>BBa_J23101</partinfo> || 35.93 <br> ± <br> 0.62 || 82.68 <br> ± <br> 1.84 || 86.42 <br> ± <br> 1.91<br />
|-<br />
|<partinfo>BBa_J23105</partinfo> || 29.86 <br> ± <br> 0.33 || 63.09 <br> ± <br> 7.08 || 85.00 <br> ± <br> 5.13<br />
|-<br />
|<partinfo>BBa_J23106</partinfo> || 29.17 <br> ± <br> 0.96 || 68.11 <br> ± <br> 4.25 || 88.71 <br> ± <br> 0.90<br />
|-<br />
|<partinfo>BBa_J23110</partinfo> || 31.28 <br> ± <br> 0.42 || 67.52 <br> ± <br> 5.87 || 76.15 <br> ± <br> 2.16<br />
|-<br />
|<partinfo>BBa_J23114</partinfo> || 28.97 <br> ± <br> 0.49 || 59.44 <br> ± <br> 5.20 || 80.12 <br> ± <br> 0.95<br />
|-<br />
|<partinfo>BBa_J23116</partinfo> || 28.14 <br> ± <br> 0.25 || 72.74 <br> ± <br> 0.37 || 81.68 <br> ± <br> 3.08<br />
|-<br />
|<partinfo>BBa_J23118</partinfo> || 32.84 <br> ± <br> 0.31 || 73.64 <br> ± <br> 2.41 || 89.86 <br> ± <br> 2.93<br />
|}<br />
<br />
It was not possible to evaluate promoters activities in low copy number plasmids in LB because the RFP activity was too weak and not distinguishable from the background.<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the tested conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
<br />
<br />
=<partinfo>BBa_P1004</partinfo> - chloramphenicol resistance cassette=<br />
<br />
<partinfo>BBa_P1004</partinfo> has been successfully used in the assembly of <partinfo>BBa_K300000</partinfo> integrative base vector for ''E. coli''. All the intermediate parts which contained <partinfo>BBa_P1004</partinfo> showed Chloramphenicol resistance (tested up to 34 ug/ml in LB media) when transformed in TOP10 (<partinfo>BBa_V1009</partinfo>), DH5alpha (<partinfo>BBa_V1001</partinfo>), MC1061 (<partinfo>BBa_K300078</partinfo>), MG1655 (<partinfo>BBa_V1000</partinfo>), BW23474 (<partinfo>BBa_K300985</partinfo>) and DB3.1 (<partinfo>BBa_V1005</partinfo>) strains in a high-copy plasmid.<br />
<br />
<br />
=<partinfo>BBa_K125500</partinfo> - GFP fusion brick=<br />
This part can be useful to construct fluorescent fusion proteins. It is composed by a tail domain (the GFP <partinfo>K125500</partinfo>) with a transcriptional terminator (<partinfo>BBa_B0015</partinfo>) downstream. <br />
<br />
Other protein domains can be fused upstream of this part in order to create chimeric fluorescent proteins, or it can be ligated to tags useful for low-cost protein purification.<br />
<br />
This part was used do design the following BioBrick measurement systems:<br />
<br />
*<partinfo>BBa_K300086</partinfo><br />
*<partinfo>BBa_K300088</partinfo><br />
*<partinfo>BBa_K300090</partinfo><br />
*<partinfo>BBa_K300091</partinfo><br />
*<partinfo>BBa_K300092</partinfo><br />
*<partinfo>BBa_K300099</partinfo><br />
<br />
to test these contructs:<br />
<br />
*<partinfo>BBa_K300002</partinfo><br />
*<partinfo>BBa_K300093</partinfo><br />
*<partinfo>BBa_K300094</partinfo><br />
*<partinfo>BBa_K300095</partinfo><br />
*<partinfo>BBa_K300084</partinfo><br />
*<partinfo>BBa_K300097</partinfo><br />
<br />
respectively. All of the tested parts are synthetic fusion tags whose activity could be measured by assembling a promoter with RBS upstream and a tail domain with terminator downstream, thus yielding the measurement systems. <partinfo>BBa_K300005</partinfo> was used as a tail domain with terminator downstream. It has been assembled to test the correct folding of the resulting fusion protein by measuring the GFP and to test the affinity tag performance with a proof of concept protein.<br />
<br />
<br />
In all of the measurement parts, GFP could be successfully detected in bacteria harbouring the measurement parts in high copy plasmids. In this condition, GFP was detected by using an excitation filter at 485nm and an emission filter at 540nm in a Infinite F200 microplate reader (Tecan).<br />
<br />
<table><br />
<tr><br />
<td>[[Image:UNIPV10_pTET_c_GFP.png|thumb|300px|Raw GFP curve]]</td><br />
<td>[[Image:UNIPV10_pTET_c_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]</td><br />
</tr><br />
<tr><br />
<td>[[Image:UNIPV10_HSL_c_GFP.png|thumb|300px|Raw GFP curve]]</td><br />
<td>[[Image:UNIPV10_HSL_c_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]</td><br />
</tr><br />
</table><br />
<br />
=<partinfo>BBa_J72008</partinfo> - phi80 integration helper plasmid pInt80-649=<br />
<br />
<partinfo>BBa_J72008</partinfo> has been successfully used in the integration protocol of both MC1061 (<partinfo>BBa_K300078</partinfo>) and MG1655 (<partinfo>BBa_V1000</partinfo>) ''E. coli'' strains. See [http://partsregistry.org/Part:BBa_K300000:Experience BBa_K300000 Experience page] for details about how this plasmid was used.<br />
<br />
It can be actually cured at 37-42°C, while it can be propagated at 30°C.<br />
<br />
It actually enables the propagation of R6K (conditional replication origin) plasmids, thanks to its pir-116 gene.<br />
<br />
<tr><td><br></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewPartsTeam:UNIPV-Pavia/Parts/Characterization/NewParts2010-10-27T23:30:12Z<p>Susanna: /* BBa_K300010 - PoPS-based self-inducible device */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><td valign=top width="70%"><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px" width="100%"><br />
<html><p align="center"><font size="4"><b>NEW PARTS</b></font></p></html><hr><br />
<br />
<tr><td width="100%"><br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><br />
<td align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization]<br />
</td><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th><br />
</tr><br />
</table><br><br />
<br />
=New Parts: list=<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300000 - BioBrick integrative base vector for E. coli |BBa_K300000 - BioBrick integrative base vector for E. coli]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300001 - BioBrick integrative base vector for S. cerevisiae|BBa_K300001 - BioBrick integrative base vector for S. cerevisiae]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain|BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300010 - PoPS-based self-inducible device|BBa_K300010 - PoPS-based self-inducible device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification|BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification]]<br />
<br />
<br><br />
----<br />
<br><br />
<br />
<tr><td><br />
<br />
=<partinfo>BBa_K300000</partinfo> - BioBrick integrative base vector for ''E. coli''=<br />
<br />
==Materials and Methods==<br />
<br />
'''Plasmids and strains:''' the <partinfo>BBa_J72008</partinfo> helper plasmid was kindly given by Prof. JC Anderson (UC Berkeley). BW23474 (<partinfo>BBa_K300985</partinfo>), MC1061 (<partinfo>BBa_K300078</partinfo>) and MG1655 (<partinfo>BBa_V1000</partinfo>) E. coli strains and the pCP20 helper plasmid were purchased from the Coli Genetic Stock Center (Yale University).<br />
<br />
<br />
'''Verification primers:''' all the oligonucleotides were purchased from Primm (San Raffaele Biomedical Science Park, Milan, Italy). The P1 (<partinfo>BBa_K300975</partinfo>) and P4 (<partinfo>BBa_K300978</partinfo>) primers had already been used in [Anderson JC et al., 2010]. The P2 (<partinfo>BBa_K300976</partinfo>) and P3 (<partinfo>BBa_K300977</partinfo>) primers have been newly designed using ApE and Amplify 3X. P2 and P3 have been designed also considering the previously used verification primers P2 and P3 in the pG80ko integrative plasmid, described in [DeLoache W, 2009].<br />
<br />
The relative position of the P1, P2, P3 and P4 primers is shown in Fig.1:<br />
<br />
{|align=center<br />
|[[Image:relativeprimers.png|thumb|450px|center|Figure 1: Relative position of the verification primers. a) no integrants; b) single integrant and c) integrant with multiple tandem copies. P1/P2 and P3/P4 pairs give an amplicon when at least one copy of the vector is integrated in the Phi80 locus. P2/P3 pair show an amplicon only when multiple tandem copies occur.]]<br />
|}<br />
<br />
'''Competent cells preparation:''' all the ''E. coli'' strains were made competent following a slightly modified version of the protocol described in [Sambrook J et al., 1989]. Briefly, cells were grown to and OD600 of ~0.4-0.6, harvested (4000 rpm, 10 min, 4°C) and the supernatant discarded. Cells were resuspended in (30 ml for each 50 ml of initial culture) pre-chilled Mg-Ca buffer (80 mM MgCl2, 20 mM CaCl2), centrifuged as before and the supernatant discarded. Cells were resuspended in (2 ml for each 50 ml of initial culture) pre-chilled Ca buffer (100 mM CaCl2, 15% glycerol), aliquoted in 0.5 ml tubes and freezed immediately at -80°C. Test the transformation efficiency in Colony Forming Units (CFU)/ug of transformed DNA<br />
<br />
The Chloramphenicol concentration in plates was 34 ug/ml for the high copy plasmids, 12.5 ug/ml for the medium/low copy plasmids and 12.5 for the three control strains transformed with the R6K plasmid.<br />
<br />
<br />
'''Integration protocol:'''<br />
<br />
# Transform the <partinfo>BBa_J72008</partinfo> helper plasmid in the host strain (MC1061 or MG1655) and select transformants on Amp (50 ug/ml) plates under permissive conditions (30°C) overnight.<br />
# Inoculate a single colony in selective LB and let the culture grow at 30°C, 220 rpm. When the culture reaches the OD600 of 0.4-0.6 prepare chemically competent cells.<br />
# Transform the integrative vector with the desired insert in the BBa_J72008-containing strain and select co-transformants on Cm (34 ug/ml) plates under permissive conditions (30°C) overnight. At this temperature <partinfo>BBa_J72008</partinfo> can be replicated and so the pir protein product can be expressed in the cells. The pir product enables the propagation of the integrative vector by replicating the R6K origin.<br />
# Inoculate a single colony in 5 ml of LB + Cm at 12.5 ug/ml and incubate the culture at 37°C, 220 rpm overnight. At this temperature the <partinfo>BBa_J72008</partinfo> helper cannot be replicated and the Phi80 integrase is expressed by the remaining copies of the helper. The bacteria that are able to grow in this selective medium should be correct integrants because the integrative vector cannot be replicated by the pir product anymore.<br />
# Streak the culture on a Cm plate (at 12.5 ug/ml) and incubate it at 43°C overnight to ensure the loss of the helper plasmid. The bacteria that form colonies should be correct integrants without the <partinfo>BBa_J72008</partinfo> helper plasmid.<br />
<br />
Validate the loss of the helper plasmid by inoculating colonies in Cm (at 12.5 ug/ml) media and counterselecting them in Amp (at 50 ug/ml) media. Validate the correct integration position by performing colony PCR with primers P1/P2, P3/P4, P1/P4, P2,P3 and VF2/VR. Validate the phenotype (when possible).<br />
<br />
<br />
Expected amplicon length [bp] when the vector is integrated into the Phi80 locus:<br />
{|border=1<br />
|&nbsp;<br />
|'''No integrant'''<br />
|'''Single integrant'''<br />
|'''Multiple tandem integrants (>1)'''<br />
|-<br />
|'''VF2/VR'''<br />
|none<br />
|280 + insert length<br />
|280 + insert length<br />
|-<br />
|'''P1/P4'''<br />
|546<br />
|546 + insert length + 2171 (i.e. the BBa_K300000 length)<br />
|546 + insert length + 2171 (i.e. the BBa_K300000 length)<br />
|-<br />
|'''P1/P2'''<br />
|none<br />
|452<br />
|452<br />
|-<br />
|'''P3/P4'''<br />
|none<br />
|666<br />
|666<br />
|-<br />
|'''P2/P3'''<br />
|none<br />
|none<br />
|572<br />
|}<br />
<br />
<br />
'''Marker excision protocol:'''<br />
<br />
# Inoculate an integrant in selective LB medium and let it grow to OD600=0.4-0.6. Prepare chemically competent cells.<br />
# Transform the pCP20 helper plasmid in the competent strain and select transformants on Amp (100 ug/ml) plates under permissive conditions (30°C) overnight. At this temperature the pCP20 can be replicated. The pCP20 plasmid contains Amp and Cm resistance markers, a thermoinducible Flp recombinase expression system and a heat-sensitive replication origin. The permissive temperatures for the pCP20 propagation are the same as <partinfo>BBa_J72008</partinfo>.<br />
# Inoculate a single colony in 5 ml of LB without antibiotic and incubate the culture at 37°C, 220 rpm overnight. At this temperature the pCP20 helper cannot be replicated and the Flp recombinase is expressed by the remaining copies of the helper. The bacteria should loose the R6K origin and the Cm resistance upon FRT sites recombination, mediated by Flp.<br />
# Streak the culture on a LB plate and incubate it at 43°C overnight to ensure the loss of the helper plasmid. The bacteria that form colonies should be without the pCP20 helper plasmid.<br />
Validate the loss of the helper plasmid by inoculating colonies in Amp (at 100 ug/ml) media and validate the loss of the Cm resistance from the genome by inoculating colonies in Cm (at 12.5 ug/ml) media. Validate the correct length of the integrated part without Cm resistance and R6K origin by performing colony PCR with primers P1/P4 (which amplify the entire Phi80 locus) and VF2/VR (which amplify the integrated part). Validate the phenotype (when possible).<br />
<br />
<br />
'''Colony PCR:''' a single colony or 1 ul of culture was added to the Invitrogen Platinum Taq reaction mix and was heated at 94°C for 10 min. Then it was assayed with this cycle (X 35): 94°C 30 sec, 60°C (for VF2/VR) or 63°C (for the other primers) 30 sec, 72°C according to the amplicon expected length (1Kb/min). Then the reaction was kept at 72°C for 10 min and it was run on a 1% agarose gel with the GeneRuler 1Kb Plus DNA ladder (Fermentas).<br />
<br />
<br />
'''Fluorescence assays:''' integrants were inoculated in 1 ml of M9 + Cm (12.5 ug/ml) and grown at 37°C, 220 rpm overnight. The cultures were diluted 1:100 in 2 ml of selective M9 and let grow for about 4-6 hours under the same conditions as before. Three 200 ul aliquots for each culture were transferred to a 96-well microplate and assayed in the Infinite F200 microplate reader (Tecan) for about 20 hours with the following kinetic cycle: 37°C, 5 min sampling time, linear shaking 15 sec (amplitude=3), wait 5 sec, measure absorbance at 600nm, measure fluorescence with the proper filter (EX:nm/EM:540nm for GFP or EX:535nm/EM:620nm for RFP) with gain=70. The same protocol was followed for the MC1061 and the MG1655 non-integrant strains, which were grown in M9 without antibiotic.<br />
<br />
<br />
'''Data analysis:''' the absorbance measurements were normalized by subtracting the absorbance of the M9, while the fluorescence measurements were normalized by subtracting the fluorescence of the non-integrant strains over time. For each well, the S<sub>cell</sub> signal (proportional to the reporter protein synthesis rate per cell) was computed as (1/OD600*dXFP/dt), where OD600 is the normalized absorbance and XFP is the normalized fluorescence. The S<sub>cell</sub> signal was then averaged over time to obtain a single value for each well. Results are presented as the average S<sub>cell</sub> with their 95% confidence intervals of the mean.<br />
<br />
==Results==<br />
<br />
===Integration of the desired BioBrick part into the Phi80 genome locus===<br />
<br />
MC1061 and MG1655 were chosen as host strains for integration. <partinfo>BBa_K173001</partinfo> (constitutive strong promoter with GFPmut3) and the EcoRI-PstI fragment of <partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23101</partinfo> (here called PconRFP - constitutive strong promoter with RFP) were chosen as two proof of concept BioBrick parts to test the integration capability of the <partinfo>BBa_K300000</partinfo> vector in the Phi80 genome locus of these strains. For this reason, <partinfo>BBa_K173001</partinfo> and PconRFP were ligated in <partinfo>BBa_K300000</partinfo> (digested with EcoRI-PstI) and propagated using BW23474.<br />
The integration protocol was performed as described in the Materials and Methods section for 4 different combination:<br />
<br />
{|border=1<br />
|'''Integrant name'''<br />
|'''Strain'''<br />
|'''Insert of <partinfo>BBa_K300000</partinfo><br />
|-<br />
|MC-GFP<br />
|MC1061<br />
|<partinfo>BBa_K173001</partinfo><br />
|-<br />
|MC-RFP<br />
|MC1061<br />
|PconRFP<br />
|-<br />
|MG-GFP<br />
|MG1655<br />
|<partinfo>BBa_K173001</partinfo><br />
|-<br />
|MG-RFP<br />
|MG1655<br />
|PconRFP<br />
|}<br />
<br />
Three colonies grown after the overnight incubation at 43°C (step 5 of integration protocol) were analyzed for each plate. These 12 clones were called: MC-GFP-A,B,C , MC-RFP-A,B,C , MG-GFP-A,B,C and MG-RFP-A,B,C.<br />
<br />
<br />
'''Validation of the loss of BBa_J72008:''' all the picked colonies did not grow in Amp (50 ug/ml) media, thus validating that <partinfo>BBa_J72008</partinfo> Amp-resistant helper had been actually cured from the cells. However, one of these 12 clones (MG-GFP-A) also failed to grow in Cm (12.5 ug/ml) liquid media, probably because of a mistake in its inoculation. We decided not to consider this clone and to continue with 11 clones.<br />
<br />
<br />
'''Validation of the actual integration site:''' colony PCR was performed for all the 11 clones, using MC1061 and MG1655 without integrants as negative controls. Primer pairs P1/P2 and P3/P4 were used to validate the presence of the integrative vector in the Phi80 genomic locus, while the primer pair P2/P3 was used to validate the presence of multiple tandem integrants (see Fig.1 in Materials and Methods).<br />
<br />
<br />
{|align=center<br />
|[[Image:pv_P1P2.png|thumb|450px|center|Figure 2: colony PCR with P1/P2 on all the 11 integrant clones. The blank is the reaction mix without bacteria. Expected amplicon for correct integrants: 452 bp.]]<br />
|}<br />
<br />
{|align=center<br />
|[[Image:pv_P3P4.png|thumb|450px|center|Figure 3: colony PCR with P3/P4 on all the 11 integrant clones. The blank is the reaction mix without bacteria. Expected amplicon for correct integrants: 666 bp.]]<br />
|}<br />
<br />
{|align=center<br />
|[[Image:pv_P2P3.png|thumb|450px|center|Figure 4: colony PCR with P2/P3 on all the 11 integrant clones. The blank is the reaction mix without bacteria. The lanes with the amplicon were expected to come from bacteria with multiple tandem integrants. Expected amplicon for multiple integrants: 572 bp.]]<br />
|}<br />
<br />
<br />
PCR results with primers P1/P2 and P3/P4 showed that each clone had the correct integrant in the correct genomic position (see Materials and Methods for a list of the expected amplicon lengths). Negative controls showed no amplicons with primers P1/P2 as expected, but showed an unexpected band with P3/P4. The reason of the presence of this band was not further investigated and the results with this primer pair cannot be a useful tool for future analysis. Anyway, the P1/P2 primer pair can be sufficient to successfully validate the presence of the DNA of interest in the Phi80 genomic locus.<br />
<br />
PCR results with primers P2/P3 showed that two clones (MC-GFP-B and MC-GFP-C) were single integrants, while all the other clones were multiple tandem integrants (i.e. the Phi80 locus contained more than one copy of the DNA of interest). Negative controls showed no amplicons, as expected.<br />
<br />
<br />
'''Validation of the integrants phenotype:''' all the 11 clones were assayed as described in the Materials and Methods section. Unfortunately, the green fluorescent clones (MC-GFP-A,B,C and MG-GFP-B,C) did not show appreciable differences when compared to negative controls, most probably because the autofluorescence of the cells was too high and hid the GFP signal. For this reason, GFP clones were not considered for further analysis. Other instruments should be used to detect the GFP signal.<br />
<br />
On the other hand, RFP clones (MC-RFP-A,B,C and MG-RFP-A,B,C) all showed a higher fluorescence than the negative controls (see Fig.5). As Fig.5 show, the fluorescence of the three MG-RFP had a higher variability between clones when compared to the three MC-RFP. However, the clones were not necessarily expected to behave in the same way because all of them were multiple tandem integrants and the copy number of the PconRFP construct could be arbitrary.<br />
<br />
<br />
{|align=center<br />
|[[Image:pv_phenotypeRFPbefore.png|thumb|450px|center|Figure 5: relative RFP synthesis rate for all the RFP expressing clones. Note: as a reference, typical values of the relative RFP synthesis rate measured for PconRFP in a low copy vector (~5 plasmids per cell) are about 6-7 fold higher (data not shown).]]<br />
|}<br />
<br />
<br />
===Chloramphenicol resistance marker excision===<br />
<br />
The marker excision was performed on two of the previously validated integrant strains: MC-RFP-A and MG-RFP-A (even if they were multiple tandem integrants).<br />
<br />
The marker excision protocol was performed as described in the Materials and Methods section for both strains, here named:<br />
<br />
{|border=1<br />
|'''Original name'''<br />
|'''Name after marker excision'''<br />
|-<br />
|MC-RFP<br />
|MC-RFPflip<br />
|-<br />
|MG-RFP<br />
|MG-RFPflip<br />
|}<br />
<br />
<br />
Three colonies grown after the overnight incubation at 43°C (step 4 of marker excision protocol) were analyzed for each plate. These 6 clones were called MC-RFPflip-A,B,C and MG-FRPflip-A,B,C.<br />
<br />
<br />
'''Validation of the loss of pCP20 and the resistance marker:''' all the 6 picked colonies failed to grow on both Amp (100 ug/ml) media and Cm (12.5 ug/ml) media. They could only grow in LB without antibiotics, thus validating that the pCP20 helper had been actually cured and the R6K-CmR DNA containing the Chloramphenicol selection marker had been actually eliminated.<br />
<br />
<br />
'''Validation of the length of the integrated part:''' colony PCR was performed for all the 6 clones, using MC1061 and MG1655 without integrants as negative controls. Primer pairs VF2/VR and P1/P4 were used to validate if the ''passenger'' of interest was still present in the genome and the length of the entire Phi80 locus respectively after the marker excision.<br />
<br />
{|align=center<br />
|[[Image:pv_VF2VRintegrants.png|thumb|450px|center|Figure 6: colony PCR with VF2/VR on all the 6 flipped clones. The blank is the reaction mix without bacteria. Expected amplicon for correct insert: 1.2 Kb.]]<br />
|}<br />
<br />
{|align=center<br />
|[[Image:pv_P1P4integrants.png|thumb|450px|center|Figure 7: colony PCR with P1/P4 on all the 6 flipped clones. The blank is the reaction mix without bacteria. Expected amplicon for correct construct in the correct position: 2.3 Kb. Expected amplicon for the non-integrant strain MG1655: 546 bp.]]<br />
|}<br />
<br />
<br />
PCR results with primers VF2/VR showed that all the 6 clones still contain the ''passenger'' of interest, i.e. PconRFP, in the genome after the marker excision. The reaction blank, the MG1655 strain (neg control) and also the other samples showed some extra bands, but the ~1.2Kb amplicons of MC-RFP-A,B,C and MG-RFP-A,B,C had the correct length and was much brighter than the other bands.<br />
<br />
<br />
PCR results with primers P1/P4 (Fig.7) showed that an amplicon of ~2.3Kb was present in all but one screened clones, while the MG1655 negative control showed the expected 546bp length for a non-integrant. MC-RFPflip-C did not show the P1-P4 amplicon because the reaction failed: the tube was damaged and the reaction mix was completely evaporated at the end of the PCR program. For this reason, a PCR was performed again on this clone (Fig.8).<br />
<br />
The ~2.3Kb amplicon was consistent with a single integrant of <partinfo>BBa_K300000</partinfo>-PconRFP without the R6K-CmR DNA fragment, thus validating the successful excision of the FRT-flanked DNA fragment containing R6K-CmR and confirming that PconRFP was still present in the correct locus in single copy.<br />
<br />
<br />
{|align=center<br />
|[[Image:pv_P1P4singlecloneintegrant.png|thumb|300px|center|Figure 8: colony PCR with P1/P4 on MC-RFPflip-C clone. The blank is the reaction mix without bacteria. Expected amplicon for correct construct in the correct position: 2.3 Kb.]]<br />
|}<br />
<br />
<br />
These results showed that, even if the clones were multiple tandem integrants, they became single integrants after marker excision. This is because the Flp recombinase mediated the recombination of all the FRT sites of the multiple integrants until only a single FRT site was present in the Phi80 locus, thus leaving only the single integrant of interest without the selection marker in the genome.<br />
<br />
<br />
'''Validation of the marker-less phenotype:'''all the 6 clones were assayed as described in the Materials and Methods section. They all showed a low variability and their fluorescence was lower than their two ''parents'', i.e. MC-RFP-A for the MC1061 strains and MG-RFP-A for the MG1655 strains (see Fig.9). This result is consistent with the copy number of the PconRFP construct in the clones, in fact both MC-RFP-A and MG-RFP-A were multiple tandem integrants, while MC-RFPflip-A,B,C and MG-RFPflip-A,B,C were single integrants, as described above.<br />
<br />
All the MG-RFPflip showed a very low relative RFP synthesis rate when compared to the other strains, but the signal is systematically grater than the fluorescence of the negative control, thus validating the phenotype for the MG1655 strain. MC-RFPflip-A,B,C showed a higher fluorescence than MG-RFPflip-A,B,C.<br />
<br />
In conclusion, it has been demonstrated that, even after the marker excision process, the phenotype of the engineered cells is maintained.<br />
<br />
{|align=center<br />
|[[Image:pv_phenotypeRFP.png|thumb|450px|center|Figure 9: relative RFP synthesis rate for all the RFP-expressing clones after marker excision. In this figure, the bars corresponding to the fluorescence of the clones before marker excision is also reported to facilitate the comparison between them. Note that all the three ''flip'' clones are derived from MC-RFP-A for the MC1061 clones and from MG-RFP-A for the MG1655 clones.]]<br />
|}<br />
<br />
==Discussion==<br />
<br />
A novel integrative vector for ''E. coli'' has been successfully designed, constructed and used to integrate two proof of concept protein expression systems in two commonly used E. coli strains.<br />
<br />
The results showed that the vector is fully functional and can integrate into the correct targeted locus of the host chromosome through the Phi80 site-specific recombination system by using <partinfo>BBa_J72008</partinfo>, an existing BioBrick helper plasmid from the Registry. In most cases, the integration occurs in tandem copies, probably because of the too high Chloramphenicol concentration used during the selection of integrants, which forces multiple integration of Cm-resistant constructs. This concentration was the same used during the pSC101 low copy plasmid (~5 copies per cell) selection. In some cases, it is desirable to have a single copy of the desired BioBrick in the genome, for example when the gene dosage is important. In [Haldimann A and Wanner BL, 2001] the usage of Chloramphenicol at 6 ug/ml yielded a very high percentage of single integrants. However, when tested in our lab, the MG1655 strain could survive on LB plates with Cm at 6 ug/ml and also at 8 ug/ml. For this reason a higher concentration of Cm was chosen for selection. Further studies should investigate the optimal antibiotic concentration to yield the highest single integrants percentage as possible.<br />
<br />
<br />
The Flp/FRT mediated excision of the R6K and, most importantly, of the Cm resistance marker also worked by using the pCP20 helper plasmid. The estimated efficiency of this process was 100%. In addition, multiple tandem integrants became single integrants after the marker excision. This is because the Flp recombinase mediated the recombination of all the FRT sites of the multiple integrants until only a single FRT site was present in the Phi80 locus. The marker excision is a powerful tool to engineer microbial strains for industrial protein manufacturing because the engineered organism should not carry unsafe antibiotic resistances that may be diffused in the environment.<br />
<br />
<br />
The fluorescence phenotype confirmed the correct integration into the ''E. coli'' chromosome. As expected, in general multiple integrants showed a higher fluorescence than the single integrants.<br />
<br />
<br />
The BioBrick compatibility and the vector modularity give the possibility to the scientific community to stably engineer novel biological functions in ''E. coli'' with a very easy and user friendly methodology. A user’s handbook about the vector usage is shared in the Registry, as well as the users experiences and the compatibility information.<br />
<br />
<br />
<br />
=<partinfo>BBa_K300001</partinfo> - BioBrick integrative base vector for ''S. cerevisiae''=<br />
The integration capability of this vector has been tested in S288C ''S. cerevisiae'' strain (<partinfo>BBa_K300979</partinfo>). Here is reported the followed protocol and the obtained results.<br />
<br />
<br />
'''Protocol:'''<br />
<br />
*S288C strain (Open Biosystems) was inoculated in 5 ml of YPD from a long term 15% glycerol stock and grown for 24h (30°C, 200rpm).<br />
*The culture was diluted 1:10 in 50 ml of pre-warmed YPD in a 250 ml flask and was grown for additional 4 hours under the same conditions as before.<br />
*Cells were pelleted (4000 rpm, 5 min) and resuspended in 25 ml of deionized water.<br />
*Cells were pelleted (4000 rpm, 5 min), the supernatant was discarded and the pellet was resuspended in 1 ml of deionized water and transferred into a 1.5 ml tube.<br />
*Cells were pelleted (4000 rpm, 30 sec), the supernatant was discarded and the pellet was resuspended in deionized water to a final volume of 1 ml (vortex mix vigorously).<br />
*Three 100 ul aliquots were transferred into 1.5 ml tubes, while the remaining 600 ul of cells were not used in this protocol.<br />
*The three tubes were centrifuged (4000 rpm, 30 sec) and the supernatant discarded.<br />
*Each of the three pellets were resuspended (vortex mix vigorously) in 360 ul of transformation mix (240 ul of PEG 3350 50% w/v, 36 ul of LiAc 1.0 M, boiled salmon sperm DNA, 34 ul of linearized plasmid DNA plus water). The salmon sperm DNA was boiled for 5 min and pre-chilled before adding it in the transformation mix. The plasmid DNA was previously digested with SbfI (Fermentas), purified with the NucleoSpin Extract II kit (MN) and quantified with the NanoDrop in order to add 1 ug of DNA to the transformation mix.<br />
*The tubes were heated at 42°C for 40 min.<br />
*Cells were pelleted (4000 rpm, 30 sec), the supernatant was removed by pipetting and the pellet was gently resuspended in 1 ml of deionized water.<br />
*Cells were pelleted (4000 rpm, 30 sec), the supernatant was discarded, the pellet was resuspended in 1 ml of YPD and incubated at 30°C, 200 rpm for 3 hours.<br />
*Cells were pelleted (4000 rpm, 30 sec), resuspended in 200 ul of YPD and plated on a YPD agar plate with G418 antibiotic at 200 ug/ml.<br />
*The plates were incubated at 30°C for about 3 days until colonies appeared.<br />
<br />
<br />
The integration efficiency was estimated as the colony forming units (CFUs) yielded for each ug of DNA.<br />
<br />
<br />
Protocol references:<br />
<br />
[1] http://openwetware.org/wiki/High_Efficiency_Transformation<br />
<br />
[2] Guldener U, Heck S, Fiedler T, Beinhauer J, Hegemann JH (1996), A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Research, Vol. 24, No. 13 2519–2524.<br />
<br />
<br />
'''Results:'''<br />
<br />
The transformed inserts and their integration efficiency in S288C are listed here:<br />
<br />
{|border=1<br />
|'''SbfI-digested plasmid'''<br />
|'''ug of transformed DNA'''<br />
|'''# of colonies'''<br />
|'''Estimated integration efficiency [CFU/ug]'''<br />
|-<br />
|<partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300006</partinfo><br />
|1<br />
|1700<br />
|1.7*10^3<br />
|-<br />
|<partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300007</partinfo><br />
|1<br />
|6500<br />
|6.5*10^3<br />
|-<br />
|no DNA<br />
|0<br />
|0<br />
|0<br />
|}<br />
<br />
<br />
These results suggest that the integrative vector actually works and that the selection marker is highly specific (no colonies appeared on the "no DNA" plate).<br />
<br />
The correct phenotype of the S288C bearing these parts has still to be validated (by mOrange fluorescence measurement for the <partinfo>BBa_K300007</partinfo> part), as well as the actual integration position (by PCR).<br />
<br />
=<partinfo>BBa_K300004</partinfo> - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain=<br />
Inteins (Intervening Proteins) are sequences capable of self-exciding from a precursor protein through a process known as self-splicing, forming a peptide bond between the flanking proteins (exteins). Many so-called mini-Inteins have been engineered, whose key feature is the capability to completely release a flanking extein (the target protein) in response to a simple stimulus, either chemical or physical, with no need of expensive proteases.<br />
<br />
In literature, one mini-Intein was obtained through mutagenesis of ''Mycobacterium tuberculosis'' ''Mtu RecA'' Intein. The sequence of this Intein, referred to as ΔI-CM, allows for pH/heat-controlled C-terminal cleavage.<br />
<br />
Thanks to this feature, the ΔI-CM Intein can be fused downstream of an affinity tag and upstream of the protein coding sequence of interest in order to enable a cheap cleavage process to remove the N-terminal tag.<br />
<br />
The ΔI-CM Intein sequence was designed according to [Wood DW et al., 1999] and codon-optimized for ''E. coli'' to yield <partinfo>BBa_K300004</partinfo>. This part was designed as an internal domain (start/stop codons and it has Prefix and Suffix sequences compatible with RFC 23) in order to enable protein coding sequence assemblies to generate the desired synthetic self-cleavable affinity tags for protein purification.<br />
<br />
The optimal pH for cleavage has been reported to be 6.0, while a pH of 8.5 has been shown to inhibit the cleavage; C-terminus cleavage is also strongly affected by temperature >20°C [Wood DW et al., 1999]<br />
<br />
It was used to build together with phasins (<partinfo>BBa_K300002</partinfo> - head domain - and <partinfo>BBa_K300003</partinfo> - internal domain) and flexible protein domain linker (<partinfo>BBa_K105012</partinfo>) affinity tags that are able of self-cleaving to release the fusion protein of interest you need to purify:<br />
*<partinfo>BBa_K300095</partinfo><br />
*<partinfo>BBa_K300084</partinfo><br />
<br />
=<partinfo>BBa_K300010</partinfo> - PoPS-based self-inducible device=<br />
This is a PoPS-in/PoPS-out device.<br />
<br />
The luxR gene (<partinfo>BBa_C0062</partinfo>) is constitutively produced by the <partinfo>BBa_R0040</partinfo> promoter and it can activate the ''lux pR'' in presence of the autoinducer 3-oxo-C6-homoserine-lactone (3OC6HSL or simply HSL). The PoPS input regulates the production of luxI gene (<partinfo>BBa_C0061</partinfo>). It encodes for the LuxI enzyme, which is able to produce HSL. The produced HSL can diffuse in the growth media of the cells that express LuxI. The ''lux pR'' produces a PoPS output when HSL reaches a critical concentration.<br />
<br />
This device can be specialized by assembling a promoter upstream and a promoterless expression system with the gene of interest downstream. When a cell population expresses LuxI, the concentration of HSL is an increasing function of cell culture density and so the induction of the ''lux pR'' promoter occurs only when the cells reach a threshold density.<br />
<br />
In this way, the upstream promoter autoinduces the production of the target protein at a critical culture density, depending on the HSL synthesis rate. The HSL synthesis rate can be tuned by assembling promoters of different strengths upstream of ''luxI''.<br />
<br />
This enables the construction of a library of self-inducible devices capable of starting the target protein production at a predictable culture density.<br />
<br />
This device has been characterized in many different experimental conditions:<br />
* varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation);<br />
* varying the copy number of vectors containing Sender and Receiver circuits;<br />
* varying the growth medium (LB or M9).<br />
<br />
The results obtained are reported in the sections below.<br />
<br />
<partinfo>BBa_K300010</partinfo> was assembled downstream of the constitutive promoters reported in the table, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced amounts of LuxI protein that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformants could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluorescent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) in high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized in tables.<br />
<br />
<br />
<div align='center>Sender/Receiver devices assembled as a unique BioBrick part on the same vector</div><br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender/Receiver Device Vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center' width='80%'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing GFP were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]. An example of O.D.600 and fluorescence signals for a self-inducible device expressing GFP (<partinfo>BBa_K300026</partinfo>), as well as its Scell signal and the estimated threshold value, is reported below.<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|300px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|300px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|300px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|300px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest; it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]];<br />
*K_HSL is the HSL synthesis rate per cell; it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis to estimate the HSL synthesis rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double; it was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
<br>[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> *<br />
| 8.94 10^-17 <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> *<br />
| 7.53 10^-18 <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br>[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
<br />
<div id="box" style="width: 70%; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the algorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the O.D.start point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': a modular PoPS-based devices (<partinfo>BBa_K300010</partinfo> was designed and used to realize a library of self-inducible devices, able to start the production of the heterologous protein at a defined culture density. Other member of this promoters' family are derived from <partinfo>BBa_K300009</partinfo> (see the proper section for details).<br />
<br />
They were characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A graphical summary is reported in the figures below:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Typical ''E. coli'' growth curve with O.D.start evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Typical ''E. coli'' growth curve with O.D.start evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell, and an algorithm was proposed in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In the figures below, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid).<br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the autoinducer production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by the reported graphs. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate is an increasing function of the upstream promoter's strength.<br />
<br />
For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably because too high luxI expression levels and/or synthesis rate of HSL are injurious for the cell.<br />
<br />
The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices able to perform autoinduction at many different O.D.600 values, in any cellular growth phase.<br />
<br />
=<partinfo>BBa_K300093</partinfo>, <partinfo>BBa_K300094</partinfo>, <partinfo>BBa_K300097</partinfo>, <partinfo>BBa_K300095</partinfo> and <partinfo>BBa_K300084</partinfo> - Phasin and Intein-based tags for protein purification=<br />
<br />
These parts are built by assembling Phasins (<partinfo>BBa_K300002</partinfo> and <partinfo>BBa_K300003</partinfo>) that are able to bind to PolyHydroxyAlkanoates (PHA)<br />
granules and Intein (<partinfo>BBa_K300004</partinfo>), a sequence capable of self-exciding from a precursor protein through a process known as self-splicing. In addition a flexible linker sequence (<partinfo>BBa_K105012</partinfo>) has been used to connect these parts in order to facilitate the binding and folding of the tag and the target protein of interest. Thanks to Phasin and Intein properties these parts can be used as high-specific TAGs for low-cost protein purification.<br />
<br />
At the moment were were not able to test Intein efficiency, but we could check if they affected the right folding of the target protein: we achieved this goal through the Silver Standard Assembly by using the GFP (<partinfo>BBa_K300005</partinfo>).<br />
<br />
These parts has been characterized respectively through:<br />
*pTet costitutive promoter devices:<br />
**<partinfo>BBa_K300088</partinfo><br />
**<partinfo>BBa_K300090</partinfo><br />
**<partinfo>BBa_K300099</partinfo><br />
*3OC6HSL inducible devices:<br />
**<partinfo>BBa_K300091</partinfo><br />
**<partinfo>BBa_K300092</partinfo><br />
and compared to a positive (<partinfo>BBa_K173000</partinfo>) and negative (<partinfo>BBa_B0031</partinfo>) control.<br />
<br />
==pTet costitutive promoter devices==<br />
===Methods===<br />
Inoculum (into 5 ml LB+Amp) from glycerol stock of:<br />
*<partinfo>BBa_K300088</partinfo><br />
*<partinfo>BBa_K300090</partinfo><br />
*<partinfo>BBa_K300099</partinfo><br />
*<partinfo>BBa_K173000</partinfo> (positive control)<br />
*<partinfo>BBa_B0031</partinfo> (negative control)<br />
They were let grow ON at +37°C, 220 rpm.<br />
<br />
The following day cultures were diluted 1:100 and let grow again for about five hours at +37°C, 220 rpm.<br />
<br />
Optical density (O.D.) of each cell culture was than measured with TECAN Infinte F200. Samples were diluted in order to obtain the same O.D. equal to 0,02.<br />
<br />
Than we performed a 21 hours' experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200. Each value shown is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted; cultures were shaken for 15 seconds every five minutes.<br />
<br />
===Results===<br />
<br />
{|align="center"<br />
|[[Image:UNIPV10_pTET_newP_ASB.png|thumb|300px|Raw growth curve]] || [[Image:UNIPV10_pTET_newP_GFP.png|thumb|300px|Raw GFP curve]]<br />
|}<br />
<table align="center"><br />
<tr><br />
<td><br />
[[Image:UNIPV10_pTET_newP_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<table border="1" align="center"><br />
<tr align="center"><br />
<th>Culture</th><th>Doubling time [min.] ± std error</th><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K173000</partinfo></td><td>76.3336 ± 1.4362</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300088</partinfo></td><td>74.8806 ± 2.7699</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300090</partinfo></td><td>75.9433 ± 3.6808</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300099</partinfo></td><td>78.4634 ± 2.5622</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_B0031</partinfo></td><td>70.8421 ± 2.2181</td><br />
</tr><br />
</table><br />
<br />
===Discussion===<br />
All cell cultures showed a similar growth curve and doubling time was computed as described [[Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation|here]] in order to have informations about the burden due to synthesis of such fusion proteins. It's possible to see that all doubling time are very similar; it's possible to assert that the expression of these BioBrick parts doesn't cause abnormal stress to the cells.<br />
<br />
In GFP curve it's possible to appreciate that in <partinfo>BBa_K300088</partinfo>, <partinfo>BBa_K300090</partinfo>, <partinfo>BBa_K300099</partinfo> GFP accumulation it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. These results show the right folding of the green fluorescent protein assembled downstream of the genetic circuit.<br />
<br />
The mean protein synthesis rate was also computed over the growth exponential phase, showing again an appreciable GFP production rate that is about a half of the positive control.<br />
<br />
==3OC6HSL inducible devices==<br />
===Methods===<br />
Inoculum (into 5 ml LB+Amp) from glycerol stock of:<br />
*<partinfo>BBa_K300091</partinfo><br />
*<partinfo>BBa_K300092</partinfo><br />
*<partinfo>BBa_K173000</partinfo> (positive control)<br />
*<partinfo>BBa_B0031</partinfo> (negative control)<br />
They were let grow ON at +37°C, 220 rpm.<br />
<br />
The following day cultures were diluted 1:100 and let grow again for about five hours at +37°C, 220 rpm.<br />
<br />
Optical density (O.D.) of each cell culture was than measured with TECAN Infinte F200. Samples were diluted in order to obtain the same O.D. equal to 0,02.<br />
<br />
Than we performed a 21 hours' experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200. <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> circuits were induced 100nM with HSL directly into multiplate well. Each value shown is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted; cultures were shaken for 15 seconds every five minutes.<br />
<br />
===Results===<br />
<table align="center"><br />
<tr><br />
<td>[[Image:UNIPV10_HSL_c_ASB.png|thumb|300px|Raw growth curve]]</td><br />
<td>[[Image:UNIPV10_HSL_c_GFP.png|thumb|300px|Raw GFP curve]]</td><br />
</tr><br />
</table><br />
<table align="center"><br />
<tr><br />
<td>[[Image:UNIPV10_HSL_c_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]</td><br />
</tr><br />
</table><br />
<br />
<table border="1" align="center"><br />
<tr align="center"><br />
<th>Culture</th><th>Doubling time [min.] ± std error</th><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K173000</partinfo></td><td>76.3336 ± 1.4362</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300091</partinfo><br/>induced</td><td>121.1434 ± 7.0275</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300091</partinfo><br/>not induced</td><td>74.4267 ± 1.3696</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300092</partinfo><br/>induced</td><td>122.6088 ± 1.2785</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300092</partinfo><br/>not induced</td><td>71.5105 ± 2.7113</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_B0031</partinfo></td><td>70.8421 ± 2.2181</td><br />
</tr><br />
</table><br />
<br />
===Discussion===<br />
All cell cultures showed a similar growth curve and doubling time was computed as described [[Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation|here]] in order to have informations about the burden due to synthesis of such fusion proteins. It's possible to see that all doubling time are very similar except for induced cultures. In this case doubling time is much higher than posite control and not induced cultures; so it's possible to assert that in this case there's a kind of metabolic burden higher than in the others, maybe because of the inducible system.<br />
<br />
In GFP curve it's possible to appreciate that in induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> GFP accumulation profile it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. On the other hand not induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> show a profile very similar to the last one. These results show the right folding of the green fluorescent protein assembled downstream of the genetic circuit and that the inducible system works as expected.<br />
<br />
The mean protein synthesis rate was also computed over the growth exponential phase, showing again an GFP production rate that is different from negative control. Not induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> show a low GFP synthesis rate maybe due to 3OC6HSL inducible circuit leakage activity.<br />
<br />
</td><br />
</tr></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewPartsTeam:UNIPV-Pavia/Parts/Characterization/NewParts2010-10-27T23:06:29Z<p>Susanna: /* BBa_K300010 - PoPS-based self-inducible device */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><td valign=top width="70%"><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px" width="100%"><br />
<html><p align="center"><font size="4"><b>NEW PARTS</b></font></p></html><hr><br />
<br />
<tr><td width="100%"><br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><br />
<td align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization]<br />
</td><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th><br />
</tr><br />
</table><br><br />
<br />
=New Parts: list=<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300000 - BioBrick integrative base vector for E. coli |BBa_K300000 - BioBrick integrative base vector for E. coli]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300001 - BioBrick integrative base vector for S. cerevisiae|BBa_K300001 - BioBrick integrative base vector for S. cerevisiae]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain|BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300010 - PoPS-based self-inducible device|BBa_K300010 - PoPS-based self-inducible device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification|BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification]]<br />
<br />
<br><br />
----<br />
<br><br />
<br />
<tr><td><br />
<br />
=<partinfo>BBa_K300000</partinfo> - BioBrick integrative base vector for ''E. coli''=<br />
<br />
==Materials and Methods==<br />
<br />
'''Plasmids and strains:''' the <partinfo>BBa_J72008</partinfo> helper plasmid was kindly given by Prof. JC Anderson (UC Berkeley). BW23474 (<partinfo>BBa_K300985</partinfo>), MC1061 (<partinfo>BBa_K300078</partinfo>) and MG1655 (<partinfo>BBa_V1000</partinfo>) E. coli strains and the pCP20 helper plasmid were purchased from the Coli Genetic Stock Center (Yale University).<br />
<br />
<br />
'''Verification primers:''' all the oligonucleotides were purchased from Primm (San Raffaele Biomedical Science Park, Milan, Italy). The P1 (<partinfo>BBa_K300975</partinfo>) and P4 (<partinfo>BBa_K300978</partinfo>) primers had already been used in [Anderson JC et al., 2010]. The P2 (<partinfo>BBa_K300976</partinfo>) and P3 (<partinfo>BBa_K300977</partinfo>) primers have been newly designed using ApE and Amplify 3X. P2 and P3 have been designed also considering the previously used verification primers P2 and P3 in the pG80ko integrative plasmid, described in [DeLoache W, 2009].<br />
<br />
The relative position of the P1, P2, P3 and P4 primers is shown in Fig.1:<br />
<br />
{|align=center<br />
|[[Image:relativeprimers.png|thumb|450px|center|Figure 1: Relative position of the verification primers. a) no integrants; b) single integrant and c) integrant with multiple tandem copies. P1/P2 and P3/P4 pairs give an amplicon when at least one copy of the vector is integrated in the Phi80 locus. P2/P3 pair show an amplicon only when multiple tandem copies occur.]]<br />
|}<br />
<br />
'''Competent cells preparation:''' all the ''E. coli'' strains were made competent following a slightly modified version of the protocol described in [Sambrook J et al., 1989]. Briefly, cells were grown to and OD600 of ~0.4-0.6, harvested (4000 rpm, 10 min, 4°C) and the supernatant discarded. Cells were resuspended in (30 ml for each 50 ml of initial culture) pre-chilled Mg-Ca buffer (80 mM MgCl2, 20 mM CaCl2), centrifuged as before and the supernatant discarded. Cells were resuspended in (2 ml for each 50 ml of initial culture) pre-chilled Ca buffer (100 mM CaCl2, 15% glycerol), aliquoted in 0.5 ml tubes and freezed immediately at -80°C. Test the transformation efficiency in Colony Forming Units (CFU)/ug of transformed DNA<br />
<br />
The Chloramphenicol concentration in plates was 34 ug/ml for the high copy plasmids, 12.5 ug/ml for the medium/low copy plasmids and 12.5 for the three control strains transformed with the R6K plasmid.<br />
<br />
<br />
'''Integration protocol:'''<br />
<br />
# Transform the <partinfo>BBa_J72008</partinfo> helper plasmid in the host strain (MC1061 or MG1655) and select transformants on Amp (50 ug/ml) plates under permissive conditions (30°C) overnight.<br />
# Inoculate a single colony in selective LB and let the culture grow at 30°C, 220 rpm. When the culture reaches the OD600 of 0.4-0.6 prepare chemically competent cells.<br />
# Transform the integrative vector with the desired insert in the BBa_J72008-containing strain and select co-transformants on Cm (34 ug/ml) plates under permissive conditions (30°C) overnight. At this temperature <partinfo>BBa_J72008</partinfo> can be replicated and so the pir protein product can be expressed in the cells. The pir product enables the propagation of the integrative vector by replicating the R6K origin.<br />
# Inoculate a single colony in 5 ml of LB + Cm at 12.5 ug/ml and incubate the culture at 37°C, 220 rpm overnight. At this temperature the <partinfo>BBa_J72008</partinfo> helper cannot be replicated and the Phi80 integrase is expressed by the remaining copies of the helper. The bacteria that are able to grow in this selective medium should be correct integrants because the integrative vector cannot be replicated by the pir product anymore.<br />
# Streak the culture on a Cm plate (at 12.5 ug/ml) and incubate it at 43°C overnight to ensure the loss of the helper plasmid. The bacteria that form colonies should be correct integrants without the <partinfo>BBa_J72008</partinfo> helper plasmid.<br />
<br />
Validate the loss of the helper plasmid by inoculating colonies in Cm (at 12.5 ug/ml) media and counterselecting them in Amp (at 50 ug/ml) media. Validate the correct integration position by performing colony PCR with primers P1/P2, P3/P4, P1/P4, P2,P3 and VF2/VR. Validate the phenotype (when possible).<br />
<br />
<br />
Expected amplicon length [bp] when the vector is integrated into the Phi80 locus:<br />
{|border=1<br />
|&nbsp;<br />
|'''No integrant'''<br />
|'''Single integrant'''<br />
|'''Multiple tandem integrants (>1)'''<br />
|-<br />
|'''VF2/VR'''<br />
|none<br />
|280 + insert length<br />
|280 + insert length<br />
|-<br />
|'''P1/P4'''<br />
|546<br />
|546 + insert length + 2171 (i.e. the BBa_K300000 length)<br />
|546 + insert length + 2171 (i.e. the BBa_K300000 length)<br />
|-<br />
|'''P1/P2'''<br />
|none<br />
|452<br />
|452<br />
|-<br />
|'''P3/P4'''<br />
|none<br />
|666<br />
|666<br />
|-<br />
|'''P2/P3'''<br />
|none<br />
|none<br />
|572<br />
|}<br />
<br />
<br />
'''Marker excision protocol:'''<br />
<br />
# Inoculate an integrant in selective LB medium and let it grow to OD600=0.4-0.6. Prepare chemically competent cells.<br />
# Transform the pCP20 helper plasmid in the competent strain and select transformants on Amp (100 ug/ml) plates under permissive conditions (30°C) overnight. At this temperature the pCP20 can be replicated. The pCP20 plasmid contains Amp and Cm resistance markers, a thermoinducible Flp recombinase expression system and a heat-sensitive replication origin. The permissive temperatures for the pCP20 propagation are the same as <partinfo>BBa_J72008</partinfo>.<br />
# Inoculate a single colony in 5 ml of LB without antibiotic and incubate the culture at 37°C, 220 rpm overnight. At this temperature the pCP20 helper cannot be replicated and the Flp recombinase is expressed by the remaining copies of the helper. The bacteria should loose the R6K origin and the Cm resistance upon FRT sites recombination, mediated by Flp.<br />
# Streak the culture on a LB plate and incubate it at 43°C overnight to ensure the loss of the helper plasmid. The bacteria that form colonies should be without the pCP20 helper plasmid.<br />
Validate the loss of the helper plasmid by inoculating colonies in Amp (at 100 ug/ml) media and validate the loss of the Cm resistance from the genome by inoculating colonies in Cm (at 12.5 ug/ml) media. Validate the correct length of the integrated part without Cm resistance and R6K origin by performing colony PCR with primers P1/P4 (which amplify the entire Phi80 locus) and VF2/VR (which amplify the integrated part). Validate the phenotype (when possible).<br />
<br />
<br />
'''Colony PCR:''' a single colony or 1 ul of culture was added to the Invitrogen Platinum Taq reaction mix and was heated at 94°C for 10 min. Then it was assayed with this cycle (X 35): 94°C 30 sec, 60°C (for VF2/VR) or 63°C (for the other primers) 30 sec, 72°C according to the amplicon expected length (1Kb/min). Then the reaction was kept at 72°C for 10 min and it was run on a 1% agarose gel with the GeneRuler 1Kb Plus DNA ladder (Fermentas).<br />
<br />
<br />
'''Fluorescence assays:''' integrants were inoculated in 1 ml of M9 + Cm (12.5 ug/ml) and grown at 37°C, 220 rpm overnight. The cultures were diluted 1:100 in 2 ml of selective M9 and let grow for about 4-6 hours under the same conditions as before. Three 200 ul aliquots for each culture were transferred to a 96-well microplate and assayed in the Infinite F200 microplate reader (Tecan) for about 20 hours with the following kinetic cycle: 37°C, 5 min sampling time, linear shaking 15 sec (amplitude=3), wait 5 sec, measure absorbance at 600nm, measure fluorescence with the proper filter (EX:nm/EM:540nm for GFP or EX:535nm/EM:620nm for RFP) with gain=70. The same protocol was followed for the MC1061 and the MG1655 non-integrant strains, which were grown in M9 without antibiotic.<br />
<br />
<br />
'''Data analysis:''' the absorbance measurements were normalized by subtracting the absorbance of the M9, while the fluorescence measurements were normalized by subtracting the fluorescence of the non-integrant strains over time. For each well, the S<sub>cell</sub> signal (proportional to the reporter protein synthesis rate per cell) was computed as (1/OD600*dXFP/dt), where OD600 is the normalized absorbance and XFP is the normalized fluorescence. The S<sub>cell</sub> signal was then averaged over time to obtain a single value for each well. Results are presented as the average S<sub>cell</sub> with their 95% confidence intervals of the mean.<br />
<br />
==Results==<br />
<br />
===Integration of the desired BioBrick part into the Phi80 genome locus===<br />
<br />
MC1061 and MG1655 were chosen as host strains for integration. <partinfo>BBa_K173001</partinfo> (constitutive strong promoter with GFPmut3) and the EcoRI-PstI fragment of <partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23101</partinfo> (here called PconRFP - constitutive strong promoter with RFP) were chosen as two proof of concept BioBrick parts to test the integration capability of the <partinfo>BBa_K300000</partinfo> vector in the Phi80 genome locus of these strains. For this reason, <partinfo>BBa_K173001</partinfo> and PconRFP were ligated in <partinfo>BBa_K300000</partinfo> (digested with EcoRI-PstI) and propagated using BW23474.<br />
The integration protocol was performed as described in the Materials and Methods section for 4 different combination:<br />
<br />
{|border=1<br />
|'''Integrant name'''<br />
|'''Strain'''<br />
|'''Insert of <partinfo>BBa_K300000</partinfo><br />
|-<br />
|MC-GFP<br />
|MC1061<br />
|<partinfo>BBa_K173001</partinfo><br />
|-<br />
|MC-RFP<br />
|MC1061<br />
|PconRFP<br />
|-<br />
|MG-GFP<br />
|MG1655<br />
|<partinfo>BBa_K173001</partinfo><br />
|-<br />
|MG-RFP<br />
|MG1655<br />
|PconRFP<br />
|}<br />
<br />
Three colonies grown after the overnight incubation at 43°C (step 5 of integration protocol) were analyzed for each plate. These 12 clones were called: MC-GFP-A,B,C , MC-RFP-A,B,C , MG-GFP-A,B,C and MG-RFP-A,B,C.<br />
<br />
<br />
'''Validation of the loss of BBa_J72008:''' all the picked colonies did not grow in Amp (50 ug/ml) media, thus validating that <partinfo>BBa_J72008</partinfo> Amp-resistant helper had been actually cured from the cells. However, one of these 12 clones (MG-GFP-A) also failed to grow in Cm (12.5 ug/ml) liquid media, probably because of a mistake in its inoculation. We decided not to consider this clone and to continue with 11 clones.<br />
<br />
<br />
'''Validation of the actual integration site:''' colony PCR was performed for all the 11 clones, using MC1061 and MG1655 without integrants as negative controls. Primer pairs P1/P2 and P3/P4 were used to validate the presence of the integrative vector in the Phi80 genomic locus, while the primer pair P2/P3 was used to validate the presence of multiple tandem integrants (see Fig.1 in Materials and Methods).<br />
<br />
<br />
{|align=center<br />
|[[Image:pv_P1P2.png|thumb|450px|center|Figure 2: colony PCR with P1/P2 on all the 11 integrant clones. The blank is the reaction mix without bacteria. Expected amplicon for correct integrants: 452 bp.]]<br />
|}<br />
<br />
{|align=center<br />
|[[Image:pv_P3P4.png|thumb|450px|center|Figure 3: colony PCR with P3/P4 on all the 11 integrant clones. The blank is the reaction mix without bacteria. Expected amplicon for correct integrants: 666 bp.]]<br />
|}<br />
<br />
{|align=center<br />
|[[Image:pv_P2P3.png|thumb|450px|center|Figure 4: colony PCR with P2/P3 on all the 11 integrant clones. The blank is the reaction mix without bacteria. The lanes with the amplicon were expected to come from bacteria with multiple tandem integrants. Expected amplicon for multiple integrants: 572 bp.]]<br />
|}<br />
<br />
<br />
PCR results with primers P1/P2 and P3/P4 showed that each clone had the correct integrant in the correct genomic position (see Materials and Methods for a list of the expected amplicon lengths). Negative controls showed no amplicons with primers P1/P2 as expected, but showed an unexpected band with P3/P4. The reason of the presence of this band was not further investigated and the results with this primer pair cannot be a useful tool for future analysis. Anyway, the P1/P2 primer pair can be sufficient to successfully validate the presence of the DNA of interest in the Phi80 genomic locus.<br />
<br />
PCR results with primers P2/P3 showed that two clones (MC-GFP-B and MC-GFP-C) were single integrants, while all the other clones were multiple tandem integrants (i.e. the Phi80 locus contained more than one copy of the DNA of interest). Negative controls showed no amplicons, as expected.<br />
<br />
<br />
'''Validation of the integrants phenotype:''' all the 11 clones were assayed as described in the Materials and Methods section. Unfortunately, the green fluorescent clones (MC-GFP-A,B,C and MG-GFP-B,C) did not show appreciable differences when compared to negative controls, most probably because the autofluorescence of the cells was too high and hid the GFP signal. For this reason, GFP clones were not considered for further analysis. Other instruments should be used to detect the GFP signal.<br />
<br />
On the other hand, RFP clones (MC-RFP-A,B,C and MG-RFP-A,B,C) all showed a higher fluorescence than the negative controls (see Fig.5). As Fig.5 show, the fluorescence of the three MG-RFP had a higher variability between clones when compared to the three MC-RFP. However, the clones were not necessarily expected to behave in the same way because all of them were multiple tandem integrants and the copy number of the PconRFP construct could be arbitrary.<br />
<br />
<br />
{|align=center<br />
|[[Image:pv_phenotypeRFPbefore.png|thumb|450px|center|Figure 5: relative RFP synthesis rate for all the RFP expressing clones. Note: as a reference, typical values of the relative RFP synthesis rate measured for PconRFP in a low copy vector (~5 plasmids per cell) are about 6-7 fold higher (data not shown).]]<br />
|}<br />
<br />
<br />
===Chloramphenicol resistance marker excision===<br />
<br />
The marker excision was performed on two of the previously validated integrant strains: MC-RFP-A and MG-RFP-A (even if they were multiple tandem integrants).<br />
<br />
The marker excision protocol was performed as described in the Materials and Methods section for both strains, here named:<br />
<br />
{|border=1<br />
|'''Original name'''<br />
|'''Name after marker excision'''<br />
|-<br />
|MC-RFP<br />
|MC-RFPflip<br />
|-<br />
|MG-RFP<br />
|MG-RFPflip<br />
|}<br />
<br />
<br />
Three colonies grown after the overnight incubation at 43°C (step 4 of marker excision protocol) were analyzed for each plate. These 6 clones were called MC-RFPflip-A,B,C and MG-FRPflip-A,B,C.<br />
<br />
<br />
'''Validation of the loss of pCP20 and the resistance marker:''' all the 6 picked colonies failed to grow on both Amp (100 ug/ml) media and Cm (12.5 ug/ml) media. They could only grow in LB without antibiotics, thus validating that the pCP20 helper had been actually cured and the R6K-CmR DNA containing the Chloramphenicol selection marker had been actually eliminated.<br />
<br />
<br />
'''Validation of the length of the integrated part:''' colony PCR was performed for all the 6 clones, using MC1061 and MG1655 without integrants as negative controls. Primer pairs VF2/VR and P1/P4 were used to validate if the ''passenger'' of interest was still present in the genome and the length of the entire Phi80 locus respectively after the marker excision.<br />
<br />
{|align=center<br />
|[[Image:pv_VF2VRintegrants.png|thumb|450px|center|Figure 6: colony PCR with VF2/VR on all the 6 flipped clones. The blank is the reaction mix without bacteria. Expected amplicon for correct insert: 1.2 Kb.]]<br />
|}<br />
<br />
{|align=center<br />
|[[Image:pv_P1P4integrants.png|thumb|450px|center|Figure 7: colony PCR with P1/P4 on all the 6 flipped clones. The blank is the reaction mix without bacteria. Expected amplicon for correct construct in the correct position: 2.3 Kb. Expected amplicon for the non-integrant strain MG1655: 546 bp.]]<br />
|}<br />
<br />
<br />
PCR results with primers VF2/VR showed that all the 6 clones still contain the ''passenger'' of interest, i.e. PconRFP, in the genome after the marker excision. The reaction blank, the MG1655 strain (neg control) and also the other samples showed some extra bands, but the ~1.2Kb amplicons of MC-RFP-A,B,C and MG-RFP-A,B,C had the correct length and was much brighter than the other bands.<br />
<br />
<br />
PCR results with primers P1/P4 (Fig.7) showed that an amplicon of ~2.3Kb was present in all but one screened clones, while the MG1655 negative control showed the expected 546bp length for a non-integrant. MC-RFPflip-C did not show the P1-P4 amplicon because the reaction failed: the tube was damaged and the reaction mix was completely evaporated at the end of the PCR program. For this reason, a PCR was performed again on this clone (Fig.8).<br />
<br />
The ~2.3Kb amplicon was consistent with a single integrant of <partinfo>BBa_K300000</partinfo>-PconRFP without the R6K-CmR DNA fragment, thus validating the successful excision of the FRT-flanked DNA fragment containing R6K-CmR and confirming that PconRFP was still present in the correct locus in single copy.<br />
<br />
<br />
{|align=center<br />
|[[Image:pv_P1P4singlecloneintegrant.png|thumb|300px|center|Figure 8: colony PCR with P1/P4 on MC-RFPflip-C clone. The blank is the reaction mix without bacteria. Expected amplicon for correct construct in the correct position: 2.3 Kb.]]<br />
|}<br />
<br />
<br />
These results showed that, even if the clones were multiple tandem integrants, they became single integrants after marker excision. This is because the Flp recombinase mediated the recombination of all the FRT sites of the multiple integrants until only a single FRT site was present in the Phi80 locus, thus leaving only the single integrant of interest without the selection marker in the genome.<br />
<br />
<br />
'''Validation of the marker-less phenotype:'''all the 6 clones were assayed as described in the Materials and Methods section. They all showed a low variability and their fluorescence was lower than their two ''parents'', i.e. MC-RFP-A for the MC1061 strains and MG-RFP-A for the MG1655 strains (see Fig.9). This result is consistent with the copy number of the PconRFP construct in the clones, in fact both MC-RFP-A and MG-RFP-A were multiple tandem integrants, while MC-RFPflip-A,B,C and MG-RFPflip-A,B,C were single integrants, as described above.<br />
<br />
All the MG-RFPflip showed a very low relative RFP synthesis rate when compared to the other strains, but the signal is systematically grater than the fluorescence of the negative control, thus validating the phenotype for the MG1655 strain. MC-RFPflip-A,B,C showed a higher fluorescence than MG-RFPflip-A,B,C.<br />
<br />
In conclusion, it has been demonstrated that, even after the marker excision process, the phenotype of the engineered cells is maintained.<br />
<br />
{|align=center<br />
|[[Image:pv_phenotypeRFP.png|thumb|450px|center|Figure 9: relative RFP synthesis rate for all the RFP-expressing clones after marker excision. In this figure, the bars corresponding to the fluorescence of the clones before marker excision is also reported to facilitate the comparison between them. Note that all the three ''flip'' clones are derived from MC-RFP-A for the MC1061 clones and from MG-RFP-A for the MG1655 clones.]]<br />
|}<br />
<br />
==Discussion==<br />
<br />
A novel integrative vector for ''E. coli'' has been successfully designed, constructed and used to integrate two proof of concept protein expression systems in two commonly used E. coli strains.<br />
<br />
The results showed that the vector is fully functional and can integrate into the correct targeted locus of the host chromosome through the Phi80 site-specific recombination system by using <partinfo>BBa_J72008</partinfo>, an existing BioBrick helper plasmid from the Registry. In most cases, the integration occurs in tandem copies, probably because of the too high Chloramphenicol concentration used during the selection of integrants, which forces multiple integration of Cm-resistant constructs. This concentration was the same used during the pSC101 low copy plasmid (~5 copies per cell) selection. In some cases, it is desirable to have a single copy of the desired BioBrick in the genome, for example when the gene dosage is important. In [Haldimann A and Wanner BL, 2001] the usage of Chloramphenicol at 6 ug/ml yielded a very high percentage of single integrants. However, when tested in our lab, the MG1655 strain could survive on LB plates with Cm at 6 ug/ml and also at 8 ug/ml. For this reason a higher concentration of Cm was chosen for selection. Further studies should investigate the optimal antibiotic concentration to yield the highest single integrants percentage as possible.<br />
<br />
<br />
The Flp/FRT mediated excision of the R6K and, most importantly, of the Cm resistance marker also worked by using the pCP20 helper plasmid. The estimated efficiency of this process was 100%. In addition, multiple tandem integrants became single integrants after the marker excision. This is because the Flp recombinase mediated the recombination of all the FRT sites of the multiple integrants until only a single FRT site was present in the Phi80 locus. The marker excision is a powerful tool to engineer microbial strains for industrial protein manufacturing because the engineered organism should not carry unsafe antibiotic resistances that may be diffused in the environment.<br />
<br />
<br />
The fluorescence phenotype confirmed the correct integration into the ''E. coli'' chromosome. As expected, in general multiple integrants showed a higher fluorescence than the single integrants.<br />
<br />
<br />
The BioBrick compatibility and the vector modularity give the possibility to the scientific community to stably engineer novel biological functions in ''E. coli'' with a very easy and user friendly methodology. A user’s handbook about the vector usage is shared in the Registry, as well as the users experiences and the compatibility information.<br />
<br />
<br />
<br />
=<partinfo>BBa_K300001</partinfo> - BioBrick integrative base vector for ''S. cerevisiae''=<br />
The integration capability of this vector has been tested in S288C ''S. cerevisiae'' strain (<partinfo>BBa_K300979</partinfo>). Here is reported the followed protocol and the obtained results.<br />
<br />
<br />
'''Protocol:'''<br />
<br />
*S288C strain (Open Biosystems) was inoculated in 5 ml of YPD from a long term 15% glycerol stock and grown for 24h (30°C, 200rpm).<br />
*The culture was diluted 1:10 in 50 ml of pre-warmed YPD in a 250 ml flask and was grown for additional 4 hours under the same conditions as before.<br />
*Cells were pelleted (4000 rpm, 5 min) and resuspended in 25 ml of deionized water.<br />
*Cells were pelleted (4000 rpm, 5 min), the supernatant was discarded and the pellet was resuspended in 1 ml of deionized water and transferred into a 1.5 ml tube.<br />
*Cells were pelleted (4000 rpm, 30 sec), the supernatant was discarded and the pellet was resuspended in deionized water to a final volume of 1 ml (vortex mix vigorously).<br />
*Three 100 ul aliquots were transferred into 1.5 ml tubes, while the remaining 600 ul of cells were not used in this protocol.<br />
*The three tubes were centrifuged (4000 rpm, 30 sec) and the supernatant discarded.<br />
*Each of the three pellets were resuspended (vortex mix vigorously) in 360 ul of transformation mix (240 ul of PEG 3350 50% w/v, 36 ul of LiAc 1.0 M, boiled salmon sperm DNA, 34 ul of linearized plasmid DNA plus water). The salmon sperm DNA was boiled for 5 min and pre-chilled before adding it in the transformation mix. The plasmid DNA was previously digested with SbfI (Fermentas), purified with the NucleoSpin Extract II kit (MN) and quantified with the NanoDrop in order to add 1 ug of DNA to the transformation mix.<br />
*The tubes were heated at 42°C for 40 min.<br />
*Cells were pelleted (4000 rpm, 30 sec), the supernatant was removed by pipetting and the pellet was gently resuspended in 1 ml of deionized water.<br />
*Cells were pelleted (4000 rpm, 30 sec), the supernatant was discarded, the pellet was resuspended in 1 ml of YPD and incubated at 30°C, 200 rpm for 3 hours.<br />
*Cells were pelleted (4000 rpm, 30 sec), resuspended in 200 ul of YPD and plated on a YPD agar plate with G418 antibiotic at 200 ug/ml.<br />
*The plates were incubated at 30°C for about 3 days until colonies appeared.<br />
<br />
<br />
The integration efficiency was estimated as the colony forming units (CFUs) yielded for each ug of DNA.<br />
<br />
<br />
Protocol references:<br />
<br />
[1] http://openwetware.org/wiki/High_Efficiency_Transformation<br />
<br />
[2] Guldener U, Heck S, Fiedler T, Beinhauer J, Hegemann JH (1996), A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Research, Vol. 24, No. 13 2519–2524.<br />
<br />
<br />
'''Results:'''<br />
<br />
The transformed inserts and their integration efficiency in S288C are listed here:<br />
<br />
{|border=1<br />
|'''SbfI-digested plasmid'''<br />
|'''ug of transformed DNA'''<br />
|'''# of colonies'''<br />
|'''Estimated integration efficiency [CFU/ug]'''<br />
|-<br />
|<partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300006</partinfo><br />
|1<br />
|1700<br />
|1.7*10^3<br />
|-<br />
|<partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300007</partinfo><br />
|1<br />
|6500<br />
|6.5*10^3<br />
|-<br />
|no DNA<br />
|0<br />
|0<br />
|0<br />
|}<br />
<br />
<br />
These results suggest that the integrative vector actually works and that the selection marker is highly specific (no colonies appeared on the "no DNA" plate).<br />
<br />
The correct phenotype of the S288C bearing these parts has still to be validated (by mOrange fluorescence measurement for the <partinfo>BBa_K300007</partinfo> part), as well as the actual integration position (by PCR).<br />
<br />
=<partinfo>BBa_K300004</partinfo> - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain=<br />
Inteins (Intervening Proteins) are sequences capable of self-exciding from a precursor protein through a process known as self-splicing, forming a peptide bond between the flanking proteins (exteins). Many so-called mini-Inteins have been engineered, whose key feature is the capability to completely release a flanking extein (the target protein) in response to a simple stimulus, either chemical or physical, with no need of expensive proteases.<br />
<br />
In literature, one mini-Intein was obtained through mutagenesis of ''Mycobacterium tuberculosis'' ''Mtu RecA'' Intein. The sequence of this Intein, referred to as ΔI-CM, allows for pH/heat-controlled C-terminal cleavage.<br />
<br />
Thanks to this feature, the ΔI-CM Intein can be fused downstream of an affinity tag and upstream of the protein coding sequence of interest in order to enable a cheap cleavage process to remove the N-terminal tag.<br />
<br />
The ΔI-CM Intein sequence was designed according to [Wood DW et al., 1999] and codon-optimized for ''E. coli'' to yield <partinfo>BBa_K300004</partinfo>. This part was designed as an internal domain (start/stop codons and it has Prefix and Suffix sequences compatible with RFC 23) in order to enable protein coding sequence assemblies to generate the desired synthetic self-cleavable affinity tags for protein purification.<br />
<br />
The optimal pH for cleavage has been reported to be 6.0, while a pH of 8.5 has been shown to inhibit the cleavage; C-terminus cleavage is also strongly affected by temperature >20°C [Wood DW et al., 1999]<br />
<br />
It was used to build together with phasins (<partinfo>BBa_K300002</partinfo> - head domain - and <partinfo>BBa_K300003</partinfo> - internal domain) and flexible protein domain linker (<partinfo>BBa_K105012</partinfo>) affinity tags that are able of self-cleaving to release the fusion protein of interest you need to purify:<br />
*<partinfo>BBa_K300095</partinfo><br />
*<partinfo>BBa_K300084</partinfo><br />
<br />
=<partinfo>BBa_K300010</partinfo> - PoPS-based self-inducible device=<br />
This is a PoPS-in/PoPS-out device.<br />
<br />
The luxR gene (<partinfo>BBa_C0062</partinfo>) is constitutively produced by the <partinfo>BBa_R0040</partinfo> promoter and it can activate the ''lux pR'' in presence of the autoinducer 3-oxo-C6-homoserine-lactone (3OC6HSL or simply HSL). The PoPS input regulates the production of luxI gene (<partinfo>BBa_C0061</partinfo>). It encodes for the LuxI enzyme, which is able to produce HSL. The produced HSL can diffuse in the growth media of the cells that express LuxI. The ''lux pR'' produces a PoPS output when HSL reaches a critical concentration.<br />
<br />
This device can be specialized by assembling a promoter upstream and a promoterless expression system with the gene of interest downstream. When a cell population expresses LuxI, the concentration of HSL is an increasing function of cell culture density and so the induction of the ''lux pR'' promoter occurs only when the cells reach a threshold density.<br />
<br />
In this way, the upstream promoter autoinduces the production of the target protein at a critical culture density, depending on the HSL synthesis rate. The HSL synthesis rate can be tuned by assembling promoters of different strengths upstream of ''luxI''.<br />
<br />
This enables the construction of a library of self-inducible devices capable of starting the target protein production at a predictable culture density.<br />
<br />
<br />
Source<br />
Receivers and Senders Registry parts.<br />
<br />
Design notes<br />
We used BioBrick Standard Assembly.<br />
<br />
<br />
This device has been characterized in many different experimental conditions:<br />
* varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation);<br />
* varying the copy number of vectors containing Sender and Receiver circuits;<br />
* varying the growth medium (LB or M9).<br />
<br />
The results obtained are reported in the sections below.<br />
<br />
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^<br />
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^<br />
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^<br />
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^<br />
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^<br />
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^<br />
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^<br />
<br />
<br />
<partinfo>BBa_K300010</partinfo> was assembled downstream of the constitutive promoters reported in the table, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced amounts of LuxI protein that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformants could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluorescent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) in high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized in tables.<br />
<br />
<br />
<div align='center>Sender/Receiver devices assembled as a unique BioBrick part on the same vector</div><br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender/Receiver Device Vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center' width='80%'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing GFP were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]. An example of O.D.600 and fluorescence signals for a self-inducible device expressing GFP (<partinfo>BBa_K300026</partinfo>), as well as its Scell signal and the estimated threshold value, is reported below.<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|300px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|300px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|300px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|300px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest; it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]];<br />
*K_HSL is the HSL synthesis rate per cell; it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis to estimate the HSL synthesis rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double; it was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
<br>[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> *<br />
| 8.94 10^-17 <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> *<br />
| 7.53 10^-18 <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br>[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
<br />
<div id="box" style="width: 70%; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the algorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the O.D.start point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': a modular PoPS-based devices (<partinfo>BBa_K300010</partinfo> was designed and used to realize a library of self-inducible devices, able to start the production of the heterologous protein at a defined culture density. Other member of this promoters' family are derived from <partinfo>BBa_K300009</partinfo> (see the proper section for details).<br />
<br />
They were characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A graphical summary is reported in the figures below:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Typical ''E. coli'' growth curve with O.D.start evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Typical ''E. coli'' growth curve with O.D.start evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell, and an algorithm was proposed in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In the figures below, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid).<br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the autoinducer production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by the reported graphs. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate is an increasing function of the upstream promoter's strength.<br />
<br />
For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably because too high luxI expression levels and/or synthesis rate of HSL are injurious for the cell.<br />
<br />
The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices able to perform autoinduction at many different O.D.600 values, in any cellular growth phase.<br />
<br />
=<partinfo>BBa_K300093</partinfo>, <partinfo>BBa_K300094</partinfo>, <partinfo>BBa_K300097</partinfo>, <partinfo>BBa_K300095</partinfo> and <partinfo>BBa_K300084</partinfo> - Phasin and Intein-based tags for protein purification=<br />
<br />
These parts are built by assembling Phasins (<partinfo>BBa_K300002</partinfo> and <partinfo>BBa_K300003</partinfo>) that are able to bind to PolyHydroxyAlkanoates (PHA)<br />
granules and Intein (<partinfo>BBa_K300004</partinfo>), a sequence capable of self-exciding from a precursor protein through a process known as self-splicing. In addition a flexible linker sequence (<partinfo>BBa_K105012</partinfo>) has been used to connect these parts in order to facilitate the binding and folding of the tag and the target protein of interest. Thanks to Phasin and Intein properties these parts can be used as high-specific TAGs for low-cost protein purification.<br />
<br />
At the moment were were not able to test Intein efficiency, but we could check if they affected the right folding of the target protein: we achieved this goal through the Silver Standard Assembly by using the GFP (<partinfo>BBa_K300005</partinfo>).<br />
<br />
These parts has been characterized respectively through:<br />
*pTet costitutive promoter devices:<br />
**<partinfo>BBa_K300088</partinfo><br />
**<partinfo>BBa_K300090</partinfo><br />
**<partinfo>BBa_K300099</partinfo><br />
*3OC6HSL inducible devices:<br />
**<partinfo>BBa_K300091</partinfo><br />
**<partinfo>BBa_K300092</partinfo><br />
and compared to a positive (<partinfo>BBa_K173000</partinfo>) and negative (<partinfo>BBa_B0031</partinfo>) control.<br />
<br />
==pTet costitutive promoter devices==<br />
===Methods===<br />
Inoculum (into 5 ml LB+Amp) from glycerol stock of:<br />
*<partinfo>BBa_K300088</partinfo><br />
*<partinfo>BBa_K300090</partinfo><br />
*<partinfo>BBa_K300099</partinfo><br />
*<partinfo>BBa_K173000</partinfo> (positive control)<br />
*<partinfo>BBa_B0031</partinfo> (negative control)<br />
They were let grow ON at +37°C, 220 rpm.<br />
<br />
The following day cultures were diluted 1:100 and let grow again for about five hours at +37°C, 220 rpm.<br />
<br />
Optical density (O.D.) of each cell culture was than measured with TECAN Infinte F200. Samples were diluted in order to obtain the same O.D. equal to 0,02.<br />
<br />
Than we performed a 21 hours' experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200. Each value shown is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted; cultures were shaken for 15 seconds every five minutes.<br />
<br />
===Results===<br />
<br />
{|align="center"<br />
|[[Image:UNIPV10_pTET_newP_ASB.png|thumb|300px|Raw growth curve]] || [[Image:UNIPV10_pTET_newP_GFP.png|thumb|300px|Raw GFP curve]]<br />
|}<br />
<table align="center"><br />
<tr><br />
<td><br />
[[Image:UNIPV10_pTET_newP_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<table border="1" align="center"><br />
<tr align="center"><br />
<th>Culture</th><th>Doubling time [min.] ± std error</th><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K173000</partinfo></td><td>76.3336 ± 1.4362</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300088</partinfo></td><td>74.8806 ± 2.7699</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300090</partinfo></td><td>75.9433 ± 3.6808</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300099</partinfo></td><td>78.4634 ± 2.5622</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_B0031</partinfo></td><td>70.8421 ± 2.2181</td><br />
</tr><br />
</table><br />
<br />
===Discussion===<br />
All cell cultures showed a similar growth curve and doubling time was computed as described [[Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation|here]] in order to have informations about the burden due to synthesis of such fusion proteins. It's possible to see that all doubling time are very similar; it's possible to assert that the expression of these BioBrick parts doesn't cause abnormal stress to the cells.<br />
<br />
In GFP curve it's possible to appreciate that in <partinfo>BBa_K300088</partinfo>, <partinfo>BBa_K300090</partinfo>, <partinfo>BBa_K300099</partinfo> GFP accumulation it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. These results show the right folding of the green fluorescent protein assembled downstream of the genetic circuit.<br />
<br />
The mean protein synthesis rate was also computed over the growth exponential phase, showing again an appreciable GFP production rate that is about a half of the positive control.<br />
<br />
==3OC6HSL inducible devices==<br />
===Methods===<br />
Inoculum (into 5 ml LB+Amp) from glycerol stock of:<br />
*<partinfo>BBa_K300091</partinfo><br />
*<partinfo>BBa_K300092</partinfo><br />
*<partinfo>BBa_K173000</partinfo> (positive control)<br />
*<partinfo>BBa_B0031</partinfo> (negative control)<br />
They were let grow ON at +37°C, 220 rpm.<br />
<br />
The following day cultures were diluted 1:100 and let grow again for about five hours at +37°C, 220 rpm.<br />
<br />
Optical density (O.D.) of each cell culture was than measured with TECAN Infinte F200. Samples were diluted in order to obtain the same O.D. equal to 0,02.<br />
<br />
Than we performed a 21 hours' experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200. <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> circuits were induced 100nM with HSL directly into multiplate well. Each value shown is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted; cultures were shaken for 15 seconds every five minutes.<br />
<br />
===Results===<br />
<table align="center"><br />
<tr><br />
<td>[[Image:UNIPV10_HSL_c_ASB.png|thumb|300px|Raw growth curve]]</td><br />
<td>[[Image:UNIPV10_HSL_c_GFP.png|thumb|300px|Raw GFP curve]]</td><br />
</tr><br />
</table><br />
<table align="center"><br />
<tr><br />
<td>[[Image:UNIPV10_HSL_c_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]</td><br />
</tr><br />
</table><br />
<br />
<table border="1" align="center"><br />
<tr align="center"><br />
<th>Culture</th><th>Doubling time [min.] ± std error</th><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K173000</partinfo></td><td>76.3336 ± 1.4362</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300091</partinfo><br/>induced</td><td>121.1434 ± 7.0275</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300091</partinfo><br/>not induced</td><td>74.4267 ± 1.3696</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300092</partinfo><br/>induced</td><td>122.6088 ± 1.2785</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300092</partinfo><br/>not induced</td><td>71.5105 ± 2.7113</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_B0031</partinfo></td><td>70.8421 ± 2.2181</td><br />
</tr><br />
</table><br />
<br />
===Discussion===<br />
All cell cultures showed a similar growth curve and doubling time was computed as described [[Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation|here]] in order to have informations about the burden due to synthesis of such fusion proteins. It's possible to see that all doubling time are very similar except for induced cultures. In this case doubling time is much higher than posite control and not induced cultures; so it's possible to assert that in this case there's a kind of metabolic burden higher than in the others, maybe because of the inducible system.<br />
<br />
In GFP curve it's possible to appreciate that in induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> GFP accumulation profile it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. On the other hand not induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> show a profile very similar to the last one. These results show the right folding of the green fluorescent protein assembled downstream of the genetic circuit and that the inducible system works as expected.<br />
<br />
The mean protein synthesis rate was also computed over the growth exponential phase, showing again an GFP production rate that is different from negative control. Not induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> show a low GFP synthesis rate maybe due to 3OC6HSL inducible circuit leakage activity.<br />
<br />
</td><br />
</tr></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistryTeam:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry2010-10-27T21:48:52Z<p>Susanna: /* BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><td valign=top width="70%"><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<html><p align="center"><font size="4"><b>EXISTING PARTS FROM THE REGISTRY</b></font></p></html><hr><br />
<br />
<tr><td width="100%"><br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><br />
<td align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization]<br />
</td><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th><br />
</tr><br />
</table><br><br />
<br />
=Existing Parts from the Registry: list=<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - R6K Origin of replication|BBa_J61001 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J2311 - constitutive promoters from Anderson's collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - chloramphenicol resistance cassette|BBa_P1004 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - GFP fusion brick|BBa_K125500 - GFP fusion brick]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=<partinfo>BBa_K300009</partinfo>/<partinfo>BBa_I4102</partinfo> - PoPS->3OC6HSL sender device=<br />
=<partinfo>BBa_F2620</partinfo> - 3OC6HSL -> PoPS Receiver=<br />
=<partinfo>BBa_J61001</partinfo> - R6K Origin of replication=<br />
<br />
=<partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> - constitutive promoters from Anderson's collection=<br />
<br />
The <partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> were charcterized in LB and M9 supplemented with glycerol (0.4%) growth media in high copy and low copy vectors in ''E. coli'' TOP10 (<partinfo>BBa_V1009</partinfo>).<br />
<br />
RPU and doubling time were characterized for all of them, according to the protocols reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|this section]]. <br />
<br />
The following measurement systems were used for high copy plasmids:<br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23100</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23101</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23105</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23106</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23110</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23114</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23116</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23118</partinfo><br />
<br />
In order to build low copy plasmid measurement systems, the EcoRI-PstI fragment (J231xx-RFP) of each <partinfo>BBa_J61002</partinfo>-BBa_J231xx was assembled into <partinfo>pSB4C5</partinfo> vector. This fragment contains the constitutive promoter of interest upstream a RBS-RFP-TT expression system.<br />
<br />
The following measurement parts were used for low copy plasmids:<br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23100</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23101</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23105</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23106</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23110</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23114</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23116</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23118</partinfo><br />
<br />
<br />
The RPU values and doubling times are here reported:<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI fragment of <partinfo>BBa_J61002</partinfo>-BBa_J231xx in <partinfo>pSB4C5</partinfo> vector, in order to transfer the promoter and the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
The error bars represent the standard deviation for three dfferent wells in the same experiment.<br />
Doubling times were evaluated for the described cultures (HC stands for High Copy and LC stands for Low Copy):<br />
{| align='center' border='1'<br />
|rowspan='2'|<b>Promoter</b><br />
|colspan='3'| <b>doubling time [minutes]</b><br />
|-<br />
| LB in HC plasmid || M9 in HC plasmid || M9 in LC plasmid<br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 33.75 <br> ± <br> 1.34 || 82.53 <br> ± <br> 2.45 || 86.11 <br> ± <br> 4.45<br />
|-<br />
|<partinfo>BBa_J23101</partinfo> || 35.93 <br> ± <br> 0.62 || 82.68 <br> ± <br> 1.84 || 86.42 <br> ± <br> 1.91<br />
|-<br />
|<partinfo>BBa_J23105</partinfo> || 29.86 <br> ± <br> 0.33 || 63.09 <br> ± <br> 7.08 || 85.00 <br> ± <br> 5.13<br />
|-<br />
|<partinfo>BBa_J23106</partinfo> || 29.17 <br> ± <br> 0.96 || 68.11 <br> ± <br> 4.25 || 88.71 <br> ± <br> 0.90<br />
|-<br />
|<partinfo>BBa_J23110</partinfo> || 31.28 <br> ± <br> 0.42 || 67.52 <br> ± <br> 5.87 || 76.15 <br> ± <br> 2.16<br />
|-<br />
|<partinfo>BBa_J23114</partinfo> || 28.97 <br> ± <br> 0.49 || 59.44 <br> ± <br> 5.20 || 80.12 <br> ± <br> 0.95<br />
|-<br />
|<partinfo>BBa_J23116</partinfo> || 28.14 <br> ± <br> 0.25 || 72.74 <br> ± <br> 0.37 || 81.68 <br> ± <br> 3.08<br />
|-<br />
|<partinfo>BBa_J23118</partinfo> || 32.84 <br> ± <br> 0.31 || 73.64 <br> ± <br> 2.41 || 89.86 <br> ± <br> 2.93<br />
|}<br />
<br />
It was not possible to evaluate promoters activities in low copy number plasmids in LB because the RFP activity was too weak and not distinguishable from the background.<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the tested conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
<br />
<br />
=<partinfo>BBa_P1004</partinfo> - chloramphenicol resistance cassette=<br />
<br />
=<partinfo>BBa_K125500</partinfo> - GFP fusion brick=<br />
This part can be useful to construct fluorescent fusion proteins. It is composed by a tail domain (the GFP <partinfo>K125500</partinfo>) with a transcriptional terminator (<partinfo>BBa_B0015</partinfo>) downstream. <br />
<br />
Other protein domains can be fused upstream of this part in order to create chimeric fluorescent proteins, or it can be ligated to tags useful for low-cost protein purification.<br />
<br />
This part was used do design the following BioBrick measurement systems:<br />
<br />
*<partinfo>BBa_K300086</partinfo><br />
*<partinfo>BBa_K300088</partinfo><br />
*<partinfo>BBa_K300090</partinfo><br />
*<partinfo>BBa_K300091</partinfo><br />
*<partinfo>BBa_K300092</partinfo><br />
*<partinfo>BBa_K300099</partinfo><br />
<br />
to test these contructs:<br />
<br />
*<partinfo>BBa_K300002</partinfo><br />
*<partinfo>BBa_K300093</partinfo><br />
*<partinfo>BBa_K300094</partinfo><br />
*<partinfo>BBa_K300095</partinfo><br />
*<partinfo>BBa_K300084</partinfo><br />
*<partinfo>BBa_K300097</partinfo><br />
<br />
respectively. All of the tested parts are synthetic fusion tags whose activity could be measured by assembling a promoter with RBS upstream and a tail domain with terminator downstream, thus yielding the measurement systems. <partinfo>BBa_K300005</partinfo> was used as a tail domain with terminator downstream. It has been assembled to test the correct folding of the resulting fusion protein by measuring the GFP and to test the affinity tag performance with a proof of concept protein.<br />
<br />
<br />
In all of the measurement parts, GFP could be successfully detected in bacteria harbouring the measurement parts in high copy plasmids. In this condition, GFP was detected by using an excitation filter at 485nm and an emission filter at 540nm in a Infinite F200 microplate reader (Tecan).<br />
<br />
<table><br />
<tr><br />
<td>[[Image:UNIPV10_pTET_c_GFP.png|thumb|300px|Raw GFP curve]]</td><br />
<td>[[Image:UNIPV10_pTET_c_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]</td><br />
</tr><br />
<tr><br />
<td>[[Image:UNIPV10_HSL_c_GFP.png|thumb|300px|Raw GFP curve]]</td><br />
<td>[[Image:UNIPV10_HSL_c_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]</td><br />
</tr><br />
</table><br />
<br />
=<partinfo>BBa_J72008</partinfo> - phi80 integration helper plasmid pInt80-649=<br />
<br />
<tr><td><br></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistryTeam:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry2010-10-27T21:36:44Z<p>Susanna: /* BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><td valign=top width="70%"><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<html><p align="center"><font size="4"><b>EXISTING PARTS FROM THE REGISTRY</b></font></p></html><hr><br />
<br />
<tr><td width="100%"><br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><br />
<td align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization]<br />
</td><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th><br />
</tr><br />
</table><br><br />
<br />
=Existing Parts from the Registry: list=<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - R6K Origin of replication|BBa_J61001 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J2311 - constitutive promoters from Anderson's collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - chloramphenicol resistance cassette|BBa_P1004 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - GFP fusion brick|BBa_K125500 - GFP fusion brick]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=<partinfo>BBa_K300009</partinfo>/<partinfo>BBa_I4102</partinfo> - PoPS->3OC6HSL sender device=<br />
=<partinfo>BBa_F2620</partinfo> - 3OC6HSL -> PoPS Receiver=<br />
=<partinfo>BBa_J61001</partinfo> - R6K Origin of replication=<br />
<br />
=<partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> - constitutive promoters from Anderson's collection=<br />
<br />
The <partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> were charcterized in LB and M9 supplemented with glycerol (0.4%) growth media in high copy and low copy vectors in ''E. coli'' TOP10 (<partinfo>BBa_V1009</partinfo>).<br />
<br />
RPU and doubling time were characterized for all of them, according to the protocols reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|this section]]. <br />
<br />
The following measurement systems were used for high copy plasmids:<br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23100</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23101</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23105</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23106</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23110</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23114</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23116</partinfo><br />
*<partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23118</partinfo><br />
<br />
In order to build low copy plasmid measurement systems, the EcoRI-PstI fragment (J231xx-RFP) of each <partinfo>BBa_J61002</partinfo>-BBa_J231xx was assembled into <partinfo>pSB4C5</partinfo> vector. This fragment contains the constitutive promoter of interest upstream a RBS-RFP-TT expression system.<br />
<br />
The following measurement parts were used for low copy plasmids:<br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23100</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23101</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23105</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23106</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23110</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23114</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23116</partinfo><br />
*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J23118</partinfo><br />
<br />
<br />
The RPU values and doubling times are here reported:<br />
GRAFICI RPU<br />
The error bars represent the standard deviation for three dfferent wells in the same experiment.<br />
<br />
TABELLINA DT<br />
<br />
<br />
<br />
DISCUSSION<br />
<br />
=<partinfo>BBa_P1004</partinfo> - chloramphenicol resistance cassette=<br />
<br />
=<partinfo>BBa_K125500</partinfo> - GFP fusion brick=<br />
This part can be useful to construct fluorescent fusion proteins. It is composed by a tail domain (the GFP <partinfo>K125500</partinfo>) with a transcriptional terminator (<partinfo>BBa_B0015</partinfo>) downstream. <br />
<br />
Other protein domains can be fused upstream of this part in order to create chimeric fluorescent proteins, or it can be ligated to tags useful for low-cost protein purification.<br />
<br />
This part was used do design the following BioBrick measurement systems:<br />
<br />
*<partinfo>BBa_K300086</partinfo><br />
*<partinfo>BBa_K300088</partinfo><br />
*<partinfo>BBa_K300090</partinfo><br />
*<partinfo>BBa_K300091</partinfo><br />
*<partinfo>BBa_K300092</partinfo><br />
*<partinfo>BBa_K300099</partinfo><br />
<br />
to test these contructs:<br />
<br />
*<partinfo>BBa_K300002</partinfo><br />
*<partinfo>BBa_K300093</partinfo><br />
*<partinfo>BBa_K300094</partinfo><br />
*<partinfo>BBa_K300095</partinfo><br />
*<partinfo>BBa_K300084</partinfo><br />
*<partinfo>BBa_K300097</partinfo><br />
<br />
respectively. All of the tested parts are synthetic fusion tags whose activity could be measured by assembling a promoter with RBS upstream and a tail domain with terminator downstream, thus yielding the measurement systems. <partinfo>BBa_K300005</partinfo> was used as a tail domain with terminator downstream. It has been assembled to test the correct folding of the resulting fusion protein by measuring the GFP and to test the affinity tag performance with a proof of concept protein.<br />
<br />
<br />
In all of the measurement parts, GFP could be successfully detected in bacteria harbouring the measurement parts in high copy plasmids. In this condition, GFP was detected by using an excitation filter at 485nm and an emission filter at 540nm in a Infinite F200 microplate reader (Tecan).<br />
<br />
<table><br />
<tr><br />
<td>[[Image:UNIPV10_pTET_c_GFP.png|thumb|300px|Raw GFP curve]]</td><br />
<td>[[Image:UNIPV10_pTET_c_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]</td><br />
</tr><br />
<tr><br />
<td>[[Image:UNIPV10_HSL_c_GFP.png|thumb|300px|Raw GFP curve]]</td><br />
<td>[[Image:UNIPV10_HSL_c_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]</td><br />
</tr><br />
</table><br />
<br />
=<partinfo>BBa_J72008</partinfo> - phi80 integration helper plasmid pInt80-649=<br />
<br />
<tr><td><br></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/CharacterizationTeam:UNIPV-Pavia/Parts/Characterization2010-10-27T19:57:17Z<p>Susanna: /* Data analysis for self-inducible promoters (initiation-treshold determination) */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign=top><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" valign="top" width="100%"><tr><td align="justify" valign="top" style="padding:20px" width="70%" colspan="2"><br />
<html><p align="center"><font size="5"><b>CHARACTERIZATION</b></font></p></html><hr><br><br />
<br />
=Our Parts=<br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th></tr><br />
<!-- <tr><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
</tr> --><br />
</table><br><br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/NewParts |New Parts]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300000 - BioBrick integrative base vector for E. coli |BBa_K300000 - BioBrick integrative base vector for E. coli]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300001 - BioBrick integrative base vector for S. cerevisiae|BBa_K300001 - BioBrick integrative base vector for S. cerevisiae]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain|BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300010 - PoPS-based self-inducible device|BBa_K300010 - PoPS-based self-inducible device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification|BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification]]<br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts |Improved Parts]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300002 - Phasin (PhaP) - head domain|BBa_K300002 - Phasin (PhaP) - head domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300003 - Phasin (PhaP) - internal domain|BBa_K300003 - Phasin (PhaP) - internal domain]]<br />
<br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry |Existing Parts from the Registry]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - R6K Origin of replication|BBa_J61001 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - chloramphenicol resistance cassette|BBa_P1004 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - GFP fusion brick|BBa_K125500 - GFP fusion brick]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<hr><br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=Growth conditions=<br />
===Microplate reader experiments for self-inducible promoters - Protocol #1===<br />
<br />
* 8 ul of long term storage glycerol stock were inoculated in 1 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
* The grown cultures were then diluted 1:100 in 1 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.<br />
* Cultures were then pelletted (2000 rpm, 10 minutes) in order to eliminate the HSL produced during the growth.<br />
* Supernatants were discarded and the pellets were resuspended in 1 ml of LB or M9 + suitable antibiotic and transferred to a 1.5 ml tube<br />
* These cultures were diluted 1:1000 in 1 ml of LB or M9 + suitable antibiotic and aliquoted in a flat-bottom 96-well microplate in triplicate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects, see https://2009.igem.org/Team:UNIPV-Pavia/Methods_Materials/Evaporation). All the wells were filled with a 200 ul volume.<br />
* The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
** 37°C constant for all the experiment;<br />
** sampling time of 5 minutes;<br />
** fluorescence gain of 50;<br />
** O.D. filter was 600 nm;<br />
** GFP filters were 485nm (ex) / 540nm (em);<br />
** 15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
** Variable experiment duration time (from 3 to 24 hours).<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2===<br />
<br />
*8 ul of long term storage glycerol stock were inoculated in 5 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
*The grown cultures were then diluted 1:100 in 5 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.<br />
*These new cultures were diluted to an O.D.600 of 0.02 (measured with a TECAN F200 microplate reader on a 200 ul of volume per well; it is not equivalent to the 1 cm pathlength cuvette) in 2 ml (wanted final volume) LB or M9 + suitable antibiotic. In order to have the cultures at the desired O.D.600 (O.D._wanted=0.02), the following dilution was performed:<br />
[[Image:UNIPV_Pavia_OD600_dil.png|500px|center]]<br />
*These new dilutions were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects in the frame). All the wells were filled with a 200 ul volume.<br />
*The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
**37°C constant for all the experiment;<br />
**sampling time of 5 minutes;<br />
**fluorescence gain of 50 or 70;<br />
**O.D. filter was 600 nm;<br />
**GFP filters were 485nm (ex) / 540nm (em);<br />
**RFP filters were 535nm (ex) / 620nm (em);<br />
**15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
**Experiment duration time: about 6 hours.<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for 3OC6-HSL quantification by means of <partinfo>BBa_T9002</partinfo> biosensor - Protocol #3===<br />
<br />
The aim of this experiment is the quantification of the 3OC6-HSL produced by cultures harbouring plasmids derived from <partinfo>BBa_K300009</partinfo>. These plasmids are BBa_J231xx-<partinfo>BBa_K300009</partinfo> assemblies, where BBa_J231xx are Anderson Promoter Collection library members. This assembled parts are 3OC6-HSL generators and one important parameter to be evaluated is the concentration of 3OC6-HSL produced.<br />
<br />
The "3OC6-HSL generator" culture was processed as follows:<br />
*<em>Preparation of 3OC6-HSL generating cultures:</em><br />
**8 ul of BBa_J231xx-<partinfo>BBa_K300009</partinfo> long term glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours. <br />
**The grown cultures were then diluted 1:100 in 5 ml of LB supplemented medium and incubated in the same conditions as before for 6 hours.<br />
**Each falcon was pelletted (2000 rpm, 10 minutes) and supernatants were collected and filtered (0.2 um), in order to eliminate bacterial residues. These supernatants contained the 3OC6-HSL at the concentration produced by cultures. These supernatants were used to "induce" <partinfo>BBa_T9002</partinfo> cultures, in order to quantify the [HSL]. They were conserved at -20°C until the next day.<br />
*<em>Growth and preparation of the biosensor culture of <partinfo>BBa_T9002</partinfo></em><br />
**8 ul of <partinfo>BBa_T9002</partinfo> long term glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
**The grown cultures were then diluted 1:100 in 5 ml of LB supplemented medium and incubated in the same conditions as before until they reached an O.D.600 of 0.07 (measured by Tecan INFINITE F200).<br />
**the required number (see below) of 200 ul aliquots of culture were transferred in each well of a 96-well microplate.<br />
*<em>Calibration curve</em><br />
**A calibration curve was obtained, by inducing in triplicate wells of <partinfo>BBa_T9002</partinfo> with different known HSL concentrations:<br />
***10 uM<br />
***1 uM<br />
***100 nM<br />
*** 50 nM<br />
*** 10 nM<br />
*** 5 nM<br />
***2 nM<br />
***1 nM<br />
*** 0,5 nM<br />
*** 0,1 nM<br />
*** 0 M<br />
**All inductions were performed adding 2ul of "inducer solution" (3OC6-HSL, Sigma Aldrich) at the proper concentration in 200 ul of culture.<br />
*<em>HSL quantification</em><br />
**2ul of supernatants prepared as described above were used to induce 200 ul of <partinfo>BBa_T9002</partinfo> cultures in triplicate. <br />
**If the amount of 3OC6-HSL present in the supernatant was not enough concentrated to trigger the induction of <partinfo>BBa_T9002</partinfo>, a bigger amount of supernatant (X ul) was used to induce the cultures (200-X ul of cultures). Then, the calibration curve was obtained by using the same amount of <partinfo>BBa_T9002</partinfo> culture (200-X ul) induced with X ul of inducer solution at the proper concentration. To maintain the same experimental conditions, the inducer was diluted in the supernatant of a negative control (i.e., <partinfo>BBa_B0034</partinfo>).<br />
**Fluorescence and absorbance were measured after 30 min from the induction with a Tecan Infinite F200 microplate reader and, using the information provided by the calibration curve, the amount of 3OC6-HSL present in every supernatant was evaluated (each reported fluorescence value was corrected with the O.D.600 of the culture).<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for <partinfo>BBa_F2620</partinfo> - Protocol #4===<br />
<br />
This protocol is identical to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|this one]], but in addition, after cultures were transferred in the 96-well microplate, 2ul of inducer solution at the proper concentration were added to each well, thus obtaining the final desired concentration.<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=Data Analysis=<br />
<br><br />
===Preliminary remarks===<br />
*All our growth curves have been obtained subtracting for each time sample the broth O.D.600 measurement from that of the culture; broth was considered in the same conditions of the culture (i.e. induced with the same inducer concentration and supplemented with the same antibiotic of the culture).<br />
<br />
*Fluorescence signals have been obtained subtracting for each time sample the fluorescent measurement of a non-fluorescent culture from that of the target culture. The non-fluorescent culture was considered in the same conditions of the culture of interest (e.g. induced with the same inducer concentration and with the same plasmid/antibiotic resistance, but without fluorescent reporter genes). This operation allows the removal, from the target fluorescent signal, of the "self-fluorescent" component and the fluorescence signal obtained is "blanked".<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Doubling time evaluation===<br />
The natural logarithm of the growth curves (processed according to the above section) was computed and the linear phase (corresponding to the bacterial exponential growth phase) was isolated by visual inspection. Then the linear regression was performed in order to estimate the slope of the line m. Finally the doubling time was estimated as d=ln(2)/m [minutes].<br />
<br />
In the case of multiple growth curves for a strain, the mean value of the processed curves was computed for each time sample before applying the above described procedure. <br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis for self-inducible promoters (initiation-treshold determination)===<br />
<br />
The task of this analysis is the evaluation of the initiation transcription point (in terms of absorbance) for self-inducible devices. The transition O.D.600 value is named, from now on, O.D.start.<br />
Data from three indipendent wells were averaged and blanked. O.D.600 signals were blanked as described in "Preliminary Remarks" section, while the fluorescence signal was blanked with the fluorescence of <partinfo>BBa_T9002</partinfo> part, assembled in the same plasmid of the considered promoter. This operation allows the removal from the fluorescence signal of the autofluorescence component and of the “leaky” component (due to the leakage of ''lux pR'' promoter in absence of the autoinducer molecule HSL).<br />
<br />
O.D.start was evaluated by computing the ''Scell'' (GFPmut3 synthesis rate per cell) signal for the desired self-inducible promoter.<br />
<br />
Scell was obtained by computing (1/O.D.600)*dGFP/dt, where O.D.600 and GFP are the blanked absorbance and fluorescence signals.<br />
<br />
The goal is the estimation of the critical O.D.600 value (O.D.start) at which the Scell significantly increases. Because Scell is a very noisy signal, a threshold value which takes into account the noise variability was proposed and it is described below.<br />
<br />
Two different signals, measured from independent samples of the same non-fluorescent culture in the same experiment, are considered: C1 and C2. The fluorescence signal of C1 and C2 (F_C1 and F_C2 respectively) can be thought as the addition of a “real signal” and a noise component.<br />
<br />
[[Image:UNIPV_Pavia_noise1.png|200px|center]]<br />
<br />
F_C1 and F_C2 have the same expected value and the same standard deviation, since they are two independent realizations of the same aleatory process: in fact, they are two time series acquired from the same cultures in the same growth conditions by the same instrument.<br />
Noise signal was computed as:<br />
<br />
[[Image:UNIPV_Pavia_noise2.png|150px|center]]<br />
<br />
An interesting signal is the Scell of ''N'' time series. It can be evaluated as the time derivative of ''N'', divided by O.D.600 of the culture. It has the behaviour shown in figure:<br />
<br />
[[Image:UNIPV_Pavia_rumoreNoiseScell.png|600px|center|Scell Noise]]<br />
It is fair to say that the noise of Scell is bigger for low O.D.600 values and its amplitude decreases dramatically for higher O.D.600 values (e.g.: O.D.600>0.1 measured with the TECAN Infinite F200 in a 96-well microplate).<br />
<br />
The O.D.600-dependent noise model proposed for the Scell signal is:<br />
<br />
[[Image:UNIPV_Pavia_ModelNoiseScell.png|300px|center|Scell noise model]]<br />
<br />
According to the formula reported above, it is possible to obtain the sigma_bar value as follows:<br />
#multiply the Scell time series values with the corresponding O.D.600;<br />
#the sigma_bar constant value can be estimated as the standard deviation of this signal.<br />
<br />
The Scell*O.D.600 signal behaviour is reported below:<br />
<br />
[[Image:UNIPV_Pavia_rumoreNoise.png|600px|center|Measurement Noise]]<br />
<br />
As this figure shows, the noise amplitude of this signal is no more O.D.600-dependent and the sigma_bar can be obtained.<br />
<br />
<br />
The evaluation of the O.D.start can be performed supposing that the transcription starts when the signal is significantly different from the noise. A threshold on the signal amplitude was estabilished as reported here:<br />
<br />
[[Image:UNIPV_Pavia_threshold.png|300px|center|variance threshold]]<br />
<br />
In order to make the method more robust, induction was considered only when 5 consecutive values exceeded the threshold amplitude.<br />
<br />
<br />
The operation of subtracting the two signals F_C1 and F_C2 is analogous to the “blanking” operation performed on data, as described previously. For, this reason, under the hypothesis that the signals have the same variance (this is a consistent hypothesis, since they are measured by the same instrument in the same experimental conditions), this argument can be extended to the “blanked” data considered in the processing. <br />
In particular, it is evident that the "blanked" signal is affected by the same noise described above and thus, the noise of Scell signal is analogous to the one described before.<br />
Thus, this threshold was used to compute the O.D.start for the cultures. This heuristic algorithm was implemented in Matlab and analysis results are reported in the “results” section.<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis to estimate the HSL synthesis rate per cell===<br />
<br />
<br />
The autoinducer synthesys rate per cell is a very important parameter to be evaluated in quorum sensing systems. <br />
In fact the knowledge of this parameter enables the rational design of cell-communication systems, such as the self-inducible devices studied in this project.<br />
<br />
A model based approach was proposed to estimate this interesting parameter. <br />
<br />
Under the hypotheses that:<br />
* no HSL is present at the beginning of the experiment - consistent hypothesis because a growth medium washing step is always performed before the experiment start in order to remove the HSL produced until that moment,<br />
* the HSL synthesis rate is not time-dependent (cells produce HSL at the same rate in each growth phase - this hypotesis simplifies the data analysis, but it should be further validated),<br />
* the half life of the autoinducer is much longer than the experiment duration (i.e. the degradation of the molecule is negligible - this hypothesis is consistent, since no HSL-degrading enzyme is present in ''E. coli''),<br />
<br />
an Ordinary Differential Equation (ODE) model which describes the HSL production in the growth media can be written:<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula1.png|250px]]<br />
|}<br />
where:<br />
* [HSL(t)] is the concentration of HSL molecule in the growth medium;<br />
* O.D.600(t) is the growth curve of the considered culture;<br />
* N is the Colony Forming Units (CFU) per O.D.600 unit (i.e. CFU in a well = O.D.600 * N)<br />
* K_HSL is the 3OC6-HSL synthesis rate per cell<br />
<br />
<br />
The analytical solution of this system is:<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula2.png|300px]]<br />
|}<br />
K_HSL can be estimated for t=t_bar, corresponding to the transcription initiation time, as reported below.<br />
<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula3.png|250px]]<br />
|}<br />
where:<br />
* N was estimated by counting TOP10 colonies obtained by plating serial dilutions of cultures with known O.D.600.<br />
* [HSL(t_bar)] was estimated as described in the section [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis - minimum induction required to activate lux pR for BBa_F2620|Data analysis - minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>]]<br />
* the O.D.600 time series was measured in each experiment, so its time integral can be computed by trapezoidal numerical integration.<br />
<br />
N was estimated in LB and M9 media and values are reported below:<br />
<br />
{| border='1' align='center'<br />
| || &nbsp;&nbsp;&nbsp;'''LB'''&nbsp;&nbsp;&nbsp; || &nbsp;&nbsp;&nbsp;'''M9'''&nbsp;&nbsp;&nbsp;<br />
|-<br />
| &nbsp; &nbsp; &nbsp; '''N''' &nbsp; &nbsp; &nbsp;|| &nbsp;&nbsp;2,818 * 10^9 &nbsp;&nbsp;||&nbsp;&nbsp; 2,123 * 10^9 &nbsp;&nbsp;<br />
|}<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data Analysis - minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>===<br />
<br />
Cultures were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for BBa_F2620 - Protocol #4|this protocol]].<br />
<br />
Data from three indipendent wells were averaged and blanked as described in "Preliminary Remarks" section.<br />
<br />
Minimum induction required to activate ''lux pR'' was evaluated by a visual inspection of Scell signal<br />
<br />
<br />
[[Image:pv_MinimumInduction.png|330px|thumb|center|Minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>]]<br />
<br />
<br />
<div align='center'><br />
'''HSL(t_bar)''' : 0,04 nM<br />
</div><br />
HSL(t_bar) was evaluated only in M9 medium because it is a minimum autofluorescence growth medium and it provides the most accurate result in estimating a low fluorescence.<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis for RPU evaluation===<br />
<br />
The RPUs are standard units proposed by Kelly J. et al., 2009, in which the relative transcriptional strength of a promoter can be measured using a reference standard.<br />
<br />
RPUs have been computed as:<br />
[[Image:UNIPV_Pavia_RPU_formula.png|400px|center]]<br />
<br />
in which:<br />
* phi is the promoter of interest and J23101 is the reference standard promoter (taken from the Anderson Promoter Collection);<br />
* F is the blanked fluorescence of the culture, computed by subtracting for each time sample the fluorescence value of a negative control (a non-fluorescent culture). In our experiments, the TOP10 cells bearing BBa_B0032 or BBa_B0033 were usually used because they are RBSs and do not have expression systems for reporter genes;<br />
* ASB is the blanked absorbance (O.D.600) of the culture, computed as described in "Preliminary remarks" section. <br />
RPU measurement has the following advantages (under suitable conditions)<br />
* it is proportional to PoPS (Polymerase Per Second), a very important parameter that expresses the transcription rate of a promoter;<br />
* it uses a reference standard and so measurements can be compared between different laboratories. <br />
The hypotheses on which RPU theory is based can be found in Kelly J. et al., 2008, as well as all the mathematical steps. From our point of view, the main hypotheses that have to be satisfied are the following:<br />
* the reporter protein must have a half life higher than the experiment duration (we use GFPmut3 - <partinfo>BBa_E0040</partinfo> -, which has an estimated half life of at least 24 hours, or an engineered RFP - <partinfo>BBa_E1010</partinfo>, for which the half life has not been measured, but is qualitatively comparable with the GFP's);<br />
* strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in J23101 reference standard.<br />
* steady state must be valid, so (dF/dt)/ASB (proportional to the GFP synthesis rate per cell) must be constant.<br />
<br />
In order to compute the RPUs, the Scell signals ((dGFP/dt)/ASB)) of the promoter of interest and of the reference J23101 were averaged in the time interval corresponding to the exponential growth phase. The boundaries of exponential phase were identified with a visual inspection of the linear phase of the logarithmic growth curve.<br />
<br />
<br><br />
----<br />
<br><br />
<br />
</td></tr></table><br />
<br />
<!-- <td valign="top"><br />
<font class="menu"><br />
<table border="0" width="80px" align="center" class="menu"><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts |New Parts]]<hr><br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]<br><br><br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts |Improved Parts]]<hr><br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry |Existing Parts from the Registry]]<hr><br />
</td></tr></table> --></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/CharacterizationTeam:UNIPV-Pavia/Parts/Characterization2010-10-27T19:56:18Z<p>Susanna: /* Data analysis for self-inducible promoters (initiation-treshold determination) */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign=top><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" valign="top" width="100%"><tr><td align="justify" valign="top" style="padding:20px" width="70%" colspan="2"><br />
<html><p align="center"><font size="5"><b>CHARACTERIZATION</b></font></p></html><hr><br><br />
<br />
=Our Parts=<br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th></tr><br />
<!-- <tr><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
</tr> --><br />
</table><br><br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/NewParts |New Parts]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300000 - BioBrick integrative base vector for E. coli |BBa_K300000 - BioBrick integrative base vector for E. coli]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300001 - BioBrick integrative base vector for S. cerevisiae|BBa_K300001 - BioBrick integrative base vector for S. cerevisiae]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain|BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300010 - PoPS-based self-inducible device|BBa_K300010 - PoPS-based self-inducible device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification|BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification]]<br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts |Improved Parts]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300002 - Phasin (PhaP) - head domain|BBa_K300002 - Phasin (PhaP) - head domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300003 - Phasin (PhaP) - internal domain|BBa_K300003 - Phasin (PhaP) - internal domain]]<br />
<br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry |Existing Parts from the Registry]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - R6K Origin of replication|BBa_J61001 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - chloramphenicol resistance cassette|BBa_P1004 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - GFP fusion brick|BBa_K125500 - GFP fusion brick]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<hr><br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=Growth conditions=<br />
===Microplate reader experiments for self-inducible promoters - Protocol #1===<br />
<br />
* 8 ul of long term storage glycerol stock were inoculated in 1 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
* The grown cultures were then diluted 1:100 in 1 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.<br />
* Cultures were then pelletted (2000 rpm, 10 minutes) in order to eliminate the HSL produced during the growth.<br />
* Supernatants were discarded and the pellets were resuspended in 1 ml of LB or M9 + suitable antibiotic and transferred to a 1.5 ml tube<br />
* These cultures were diluted 1:1000 in 1 ml of LB or M9 + suitable antibiotic and aliquoted in a flat-bottom 96-well microplate in triplicate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects, see https://2009.igem.org/Team:UNIPV-Pavia/Methods_Materials/Evaporation). All the wells were filled with a 200 ul volume.<br />
* The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
** 37°C constant for all the experiment;<br />
** sampling time of 5 minutes;<br />
** fluorescence gain of 50;<br />
** O.D. filter was 600 nm;<br />
** GFP filters were 485nm (ex) / 540nm (em);<br />
** 15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
** Variable experiment duration time (from 3 to 24 hours).<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2===<br />
<br />
*8 ul of long term storage glycerol stock were inoculated in 5 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
*The grown cultures were then diluted 1:100 in 5 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.<br />
*These new cultures were diluted to an O.D.600 of 0.02 (measured with a TECAN F200 microplate reader on a 200 ul of volume per well; it is not equivalent to the 1 cm pathlength cuvette) in 2 ml (wanted final volume) LB or M9 + suitable antibiotic. In order to have the cultures at the desired O.D.600 (O.D._wanted=0.02), the following dilution was performed:<br />
[[Image:UNIPV_Pavia_OD600_dil.png|500px|center]]<br />
*These new dilutions were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects in the frame). All the wells were filled with a 200 ul volume.<br />
*The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
**37°C constant for all the experiment;<br />
**sampling time of 5 minutes;<br />
**fluorescence gain of 50 or 70;<br />
**O.D. filter was 600 nm;<br />
**GFP filters were 485nm (ex) / 540nm (em);<br />
**RFP filters were 535nm (ex) / 620nm (em);<br />
**15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
**Experiment duration time: about 6 hours.<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for 3OC6-HSL quantification by means of <partinfo>BBa_T9002</partinfo> biosensor - Protocol #3===<br />
<br />
The aim of this experiment is the quantification of the 3OC6-HSL produced by cultures harbouring plasmids derived from <partinfo>BBa_K300009</partinfo>. These plasmids are BBa_J231xx-<partinfo>BBa_K300009</partinfo> assemblies, where BBa_J231xx are Anderson Promoter Collection library members. This assembled parts are 3OC6-HSL generators and one important parameter to be evaluated is the concentration of 3OC6-HSL produced.<br />
<br />
The "3OC6-HSL generator" culture was processed as follows:<br />
*<em>Preparation of 3OC6-HSL generating cultures:</em><br />
**8 ul of BBa_J231xx-<partinfo>BBa_K300009</partinfo> long term glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours. <br />
**The grown cultures were then diluted 1:100 in 5 ml of LB supplemented medium and incubated in the same conditions as before for 6 hours.<br />
**Each falcon was pelletted (2000 rpm, 10 minutes) and supernatants were collected and filtered (0.2 um), in order to eliminate bacterial residues. These supernatants contained the 3OC6-HSL at the concentration produced by cultures. These supernatants were used to "induce" <partinfo>BBa_T9002</partinfo> cultures, in order to quantify the [HSL]. They were conserved at -20°C until the next day.<br />
*<em>Growth and preparation of the biosensor culture of <partinfo>BBa_T9002</partinfo></em><br />
**8 ul of <partinfo>BBa_T9002</partinfo> long term glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
**The grown cultures were then diluted 1:100 in 5 ml of LB supplemented medium and incubated in the same conditions as before until they reached an O.D.600 of 0.07 (measured by Tecan INFINITE F200).<br />
**the required number (see below) of 200 ul aliquots of culture were transferred in each well of a 96-well microplate.<br />
*<em>Calibration curve</em><br />
**A calibration curve was obtained, by inducing in triplicate wells of <partinfo>BBa_T9002</partinfo> with different known HSL concentrations:<br />
***10 uM<br />
***1 uM<br />
***100 nM<br />
*** 50 nM<br />
*** 10 nM<br />
*** 5 nM<br />
***2 nM<br />
***1 nM<br />
*** 0,5 nM<br />
*** 0,1 nM<br />
*** 0 M<br />
**All inductions were performed adding 2ul of "inducer solution" (3OC6-HSL, Sigma Aldrich) at the proper concentration in 200 ul of culture.<br />
*<em>HSL quantification</em><br />
**2ul of supernatants prepared as described above were used to induce 200 ul of <partinfo>BBa_T9002</partinfo> cultures in triplicate. <br />
**If the amount of 3OC6-HSL present in the supernatant was not enough concentrated to trigger the induction of <partinfo>BBa_T9002</partinfo>, a bigger amount of supernatant (X ul) was used to induce the cultures (200-X ul of cultures). Then, the calibration curve was obtained by using the same amount of <partinfo>BBa_T9002</partinfo> culture (200-X ul) induced with X ul of inducer solution at the proper concentration. To maintain the same experimental conditions, the inducer was diluted in the supernatant of a negative control (i.e., <partinfo>BBa_B0034</partinfo>).<br />
**Fluorescence and absorbance were measured after 30 min from the induction with a Tecan Infinite F200 microplate reader and, using the information provided by the calibration curve, the amount of 3OC6-HSL present in every supernatant was evaluated (each reported fluorescence value was corrected with the O.D.600 of the culture).<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for <partinfo>BBa_F2620</partinfo> - Protocol #4===<br />
<br />
This protocol is identical to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|this one]], but in addition, after cultures were transferred in the 96-well microplate, 2ul of inducer solution at the proper concentration were added to each well, thus obtaining the final desired concentration.<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=Data Analysis=<br />
<br><br />
===Preliminary remarks===<br />
*All our growth curves have been obtained subtracting for each time sample the broth O.D.600 measurement from that of the culture; broth was considered in the same conditions of the culture (i.e. induced with the same inducer concentration and supplemented with the same antibiotic of the culture).<br />
<br />
*Fluorescence signals have been obtained subtracting for each time sample the fluorescent measurement of a non-fluorescent culture from that of the target culture. The non-fluorescent culture was considered in the same conditions of the culture of interest (e.g. induced with the same inducer concentration and with the same plasmid/antibiotic resistance, but without fluorescent reporter genes). This operation allows the removal, from the target fluorescent signal, of the "self-fluorescent" component and the fluorescence signal obtained is "blanked".<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Doubling time evaluation===<br />
The natural logarithm of the growth curves (processed according to the above section) was computed and the linear phase (corresponding to the bacterial exponential growth phase) was isolated by visual inspection. Then the linear regression was performed in order to estimate the slope of the line m. Finally the doubling time was estimated as d=ln(2)/m [minutes].<br />
<br />
In the case of multiple growth curves for a strain, the mean value of the processed curves was computed for each time sample before applying the above described procedure. <br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis for self-inducible promoters (initiation-treshold determination)===<br />
<br />
The task of this analysis is the evaluation of the initiation transcription point (in terms of absorbance) for self-inducible devices. The transition O.D.600 value is named, from now on, O.D.start.<br />
Data from three indipendent wells were averaged and blanked. O.D.600 signals were blanked as described in "Preliminary Remarks" section, while the fluorescence signal was blanked with the fluorescence of <partinfo>BBa_T9002</partinfo> part, assembled in the same plasmid of the considered promoter. This operation allows the removal from the fluorescence signal of the autofluorescence component and of the “leaky” component (due to the leakage of ''lux pR'' promoter in absence of the autoinducer molecule HSL).<br />
<br />
O.D.start was evaluated by computing the ''Scell'' (GFPmut3 synthesis rate per cell) signal for the desired self-inducible promoter.<br />
<br />
Scell was obtained by computing (1/O.D.600)*dGFP/dt, where O.D.600 and GFP are the blanked absorbance and fluorescence signals.<br />
<br />
The goal is the estimation of the critical O.D.600 value (O.D.start) at which the Scell significantly increases. Because Scell is a very noisy signal, a threshold value which takes into account the noise variability was proposed and it is described below.<br />
<br />
Two different signals, measured from independent samples of the same non-fluorescent culture in the same experiment, are considered: C1 and C2. The fluorescence signal of C1 and C2 (F_C1 and F_C2 respectively) can be thought as the addition of a “real signal” and a noise component.<br />
<br />
[[Image:UNIPV_Pavia_noise1.png|150px|center]]<br />
<br />
F_C1 and F_C2 have the same expected value and the same standard deviation, since they are two independent realizations of the same aleatory process: in fact, they are two time series acquired from the same cultures in the same growth conditions by the same instrument.<br />
Noise signal was computed as:<br />
<br />
[[Image:UNIPV_Pavia_noise2.png|50px|center]]<br />
<br />
An interesting signal is the Scell of ''N'' time series. It can be evaluated as the time derivative of ''N'', divided by O.D.600 of the culture. It has the behaviour shown in figure:<br />
<br />
[[Image:UNIPV_Pavia_rumoreNoiseScell.png|600px|center|Scell Noise]]<br />
It is fair to say that the noise of Scell is bigger for low O.D.600 values and its amplitude decreases dramatically for higher O.D.600 values (e.g.: O.D.600>0.1 measured with the TECAN Infinite F200 in a 96-well microplate).<br />
<br />
The O.D.600-dependent noise model proposed for the Scell signal is:<br />
<br />
[[Image:UNIPV_Pavia_ModelNoiseScell.png|300px|center|Scell noise model]]<br />
<br />
According to the formula reported above, it is possible to obtain the sigma_bar value as follows:<br />
#multiply the Scell time series values with the corresponding O.D.600;<br />
#the sigma_bar constant value can be estimated as the standard deviation of this signal.<br />
<br />
The Scell*O.D.600 signal behaviour is reported below:<br />
<br />
[[Image:UNIPV_Pavia_rumoreNoise.png|600px|center|Measurement Noise]]<br />
<br />
As this figure shows, the noise amplitude of this signal is no more O.D.600-dependent and the sigma_bar can be obtained.<br />
<br />
<br />
The evaluation of the O.D.start can be performed supposing that the transcription starts when the signal is significantly different from the noise. A threshold on the signal amplitude was estabilished as reported here:<br />
<br />
[[Image:UNIPV_Pavia_threshold.png|300px|center|variance threshold]]<br />
<br />
In order to make the method more robust, induction was considered only when 5 consecutive values exceeded the threshold amplitude.<br />
<br />
<br />
The operation of subtracting the two signals F_C1 and F_C2 is analogous to the “blanking” operation performed on data, as described previously. For, this reason, under the hypothesis that the signals have the same variance (this is a consistent hypothesis, since they are measured by the same instrument in the same experimental conditions), this argument can be extended to the “blanked” data considered in the processing. <br />
In particular, it is evident that the "blanked" signal is affected by the same noise described above and thus, the noise of Scell signal is analogous to the one described before.<br />
Thus, this threshold was used to compute the O.D.start for the cultures. This heuristic algorithm was implemented in Matlab and analysis results are reported in the “results” section.<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis to estimate the HSL synthesis rate per cell===<br />
<br />
<br />
The autoinducer synthesys rate per cell is a very important parameter to be evaluated in quorum sensing systems. <br />
In fact the knowledge of this parameter enables the rational design of cell-communication systems, such as the self-inducible devices studied in this project.<br />
<br />
A model based approach was proposed to estimate this interesting parameter. <br />
<br />
Under the hypotheses that:<br />
* no HSL is present at the beginning of the experiment - consistent hypothesis because a growth medium washing step is always performed before the experiment start in order to remove the HSL produced until that moment,<br />
* the HSL synthesis rate is not time-dependent (cells produce HSL at the same rate in each growth phase - this hypotesis simplifies the data analysis, but it should be further validated),<br />
* the half life of the autoinducer is much longer than the experiment duration (i.e. the degradation of the molecule is negligible - this hypothesis is consistent, since no HSL-degrading enzyme is present in ''E. coli''),<br />
<br />
an Ordinary Differential Equation (ODE) model which describes the HSL production in the growth media can be written:<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula1.png|250px]]<br />
|}<br />
where:<br />
* [HSL(t)] is the concentration of HSL molecule in the growth medium;<br />
* O.D.600(t) is the growth curve of the considered culture;<br />
* N is the Colony Forming Units (CFU) per O.D.600 unit (i.e. CFU in a well = O.D.600 * N)<br />
* K_HSL is the 3OC6-HSL synthesis rate per cell<br />
<br />
<br />
The analytical solution of this system is:<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula2.png|300px]]<br />
|}<br />
K_HSL can be estimated for t=t_bar, corresponding to the transcription initiation time, as reported below.<br />
<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula3.png|250px]]<br />
|}<br />
where:<br />
* N was estimated by counting TOP10 colonies obtained by plating serial dilutions of cultures with known O.D.600.<br />
* [HSL(t_bar)] was estimated as described in the section [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis - minimum induction required to activate lux pR for BBa_F2620|Data analysis - minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>]]<br />
* the O.D.600 time series was measured in each experiment, so its time integral can be computed by trapezoidal numerical integration.<br />
<br />
N was estimated in LB and M9 media and values are reported below:<br />
<br />
{| border='1' align='center'<br />
| || &nbsp;&nbsp;&nbsp;'''LB'''&nbsp;&nbsp;&nbsp; || &nbsp;&nbsp;&nbsp;'''M9'''&nbsp;&nbsp;&nbsp;<br />
|-<br />
| &nbsp; &nbsp; &nbsp; '''N''' &nbsp; &nbsp; &nbsp;|| &nbsp;&nbsp;2,818 * 10^9 &nbsp;&nbsp;||&nbsp;&nbsp; 2,123 * 10^9 &nbsp;&nbsp;<br />
|}<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data Analysis - minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>===<br />
<br />
Cultures were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for BBa_F2620 - Protocol #4|this protocol]].<br />
<br />
Data from three indipendent wells were averaged and blanked as described in "Preliminary Remarks" section.<br />
<br />
Minimum induction required to activate ''lux pR'' was evaluated by a visual inspection of Scell signal<br />
<br />
<br />
[[Image:pv_MinimumInduction.png|330px|thumb|center|Minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>]]<br />
<br />
<br />
<div align='center'><br />
'''HSL(t_bar)''' : 0,04 nM<br />
</div><br />
HSL(t_bar) was evaluated only in M9 medium because it is a minimum autofluorescence growth medium and it provides the most accurate result in estimating a low fluorescence.<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis for RPU evaluation===<br />
<br />
The RPUs are standard units proposed by Kelly J. et al., 2009, in which the relative transcriptional strength of a promoter can be measured using a reference standard.<br />
<br />
RPUs have been computed as:<br />
[[Image:UNIPV_Pavia_RPU_formula.png|400px|center]]<br />
<br />
in which:<br />
* phi is the promoter of interest and J23101 is the reference standard promoter (taken from the Anderson Promoter Collection);<br />
* F is the blanked fluorescence of the culture, computed by subtracting for each time sample the fluorescence value of a negative control (a non-fluorescent culture). In our experiments, the TOP10 cells bearing BBa_B0032 or BBa_B0033 were usually used because they are RBSs and do not have expression systems for reporter genes;<br />
* ASB is the blanked absorbance (O.D.600) of the culture, computed as described in "Preliminary remarks" section. <br />
RPU measurement has the following advantages (under suitable conditions)<br />
* it is proportional to PoPS (Polymerase Per Second), a very important parameter that expresses the transcription rate of a promoter;<br />
* it uses a reference standard and so measurements can be compared between different laboratories. <br />
The hypotheses on which RPU theory is based can be found in Kelly J. et al., 2008, as well as all the mathematical steps. From our point of view, the main hypotheses that have to be satisfied are the following:<br />
* the reporter protein must have a half life higher than the experiment duration (we use GFPmut3 - <partinfo>BBa_E0040</partinfo> -, which has an estimated half life of at least 24 hours, or an engineered RFP - <partinfo>BBa_E1010</partinfo>, for which the half life has not been measured, but is qualitatively comparable with the GFP's);<br />
* strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in J23101 reference standard.<br />
* steady state must be valid, so (dF/dt)/ASB (proportional to the GFP synthesis rate per cell) must be constant.<br />
<br />
In order to compute the RPUs, the Scell signals ((dGFP/dt)/ASB)) of the promoter of interest and of the reference J23101 were averaged in the time interval corresponding to the exponential growth phase. The boundaries of exponential phase were identified with a visual inspection of the linear phase of the logarithmic growth curve.<br />
<br />
<br><br />
----<br />
<br><br />
<br />
</td></tr></table><br />
<br />
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[[Team:UNIPV-Pavia/Parts/Characterization/NewParts |New Parts]]<hr><br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
</td></tr><br />
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[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]<br><br><br />
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[[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts |Improved Parts]]<hr><br />
</td></tr><br />
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[[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry |Existing Parts from the Registry]]<hr><br />
</td></tr></table> --></div>Susannahttp://2010.igem.org/File:UNIPV_Pavia_noise1.pngFile:UNIPV Pavia noise1.png2010-10-27T19:55:44Z<p>Susanna: uploaded a new version of "Image:UNIPV Pavia noise1.png"</p>
<hr />
<div></div>Susannahttp://2010.igem.org/File:UNIPV_Pavia_noise2.pngFile:UNIPV Pavia noise2.png2010-10-27T19:55:41Z<p>Susanna: uploaded a new version of "Image:UNIPV Pavia noise2.png"</p>
<hr />
<div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewPartsTeam:UNIPV-Pavia/Parts/Characterization/NewParts2010-10-27T18:37:30Z<p>Susanna: /* BBa_K300010 - PoPS-based self-inducible device */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><td valign=top width="70%"><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px" width="100%"><br />
<html><p align="center"><font size="4"><b>NEW PARTS</b></font></p></html><hr><br />
<br />
<tr><td width="100%"><br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><br />
<td align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization]<br />
</td><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th><br />
</tr><br />
</table><br><br />
<br />
=New Parts: list=<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300000 - BioBrick integrative base vector for E. coli |BBa_K300000 - BioBrick integrative base vector for E. coli]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300001 - BioBrick integrative base vector for S. cerevisiae|BBa_K300001 - BioBrick integrative base vector for S. cerevisiae]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain|BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300010 - PoPS-based self-inducible device|BBa_K300010 - PoPS-based self-inducible device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification|BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification]]<br />
<br />
<br><br />
----<br />
<br><br />
<br />
<tr><td><br />
<br />
=<partinfo>BBa_K300000</partinfo> - BioBrick integrative base vector for ''E. coli''=<br />
<br />
==Materials and Methods==<br />
<br />
'''Plasmids and strains:''' the <partinfo>BBa_J72008</partinfo> helper plasmid was kindly given by Prof. JC Anderson (UC Berkeley). BW23474 (<partinfo>BBa_K300985</partinfo>), MC1061 (<partinfo>BBa_K300078</partinfo>) and MG1655 (<partinfo>BBa_V1000</partinfo>) E. coli strains and the pCP20 helper plasmid were purchased from the Coli Genetic Stock Center (Yale University).<br />
<br />
<br />
'''Verification primers:''' all the oligonucleotides were purchased from Primm (San Raffaele Biomedical Science Park, Milan, Italy). The P1 (<partinfo>BBa_K300975</partinfo>) and P4 (<partinfo>BBa_K300978</partinfo>) primers had already been used in [Anderson JC et al., 2010]. The P2 (<partinfo>BBa_K300976</partinfo>) and P3 (<partinfo>BBa_K300977</partinfo>) primers have been newly designed using ApE and Amplify 3X. P2 and P3 have been designed also considering the previously used verification primers P2 and P3 in the pG80ko integrative plasmid, described in [DeLoache W, 2009].<br />
<br />
The relative position of the P1, P2, P3 and P4 primers is shown in Fig.1:<br />
<br />
{|align=center<br />
|[[Image:relativeprimers.png|thumb|450px|center|Figure 1: Relative position of the verification primers. a) no integrants; b) single integrant and c) integrant with multiple tandem copies. P1/P2 and P3/P4 pairs give an amplicon when at least one copy of the vector is integrated in the Phi80 locus. P2/P3 pair show an amplicon only when multiple tandem copies occur.]]<br />
|}<br />
<br />
'''Competent cells preparation:''' all the ''E. coli'' strains were made competent following a slightly modified version of the protocol described in [Sambrook J et al., 1989]. Briefly, cells were grown to and OD600 of ~0.4-0.6, harvested (4000 rpm, 10 min, 4°C) and the supernatant discarded. Cells were resuspended in (30 ml for each 50 ml of initial culture) pre-chilled Mg-Ca buffer (80 mM MgCl2, 20 mM CaCl2), centrifuged as before and the supernatant discarded. Cells were resuspended in (2 ml for each 50 ml of initial culture) pre-chilled Ca buffer (100 mM CaCl2, 15% glycerol), aliquoted in 0.5 ml tubes and freezed immediately at -80°C. Test the transformation efficiency in Colony Forming Units (CFU)/ug of transformed DNA<br />
<br />
The Chloramphenicol concentration in plates was 34 ug/ml for the high copy plasmids, 12.5 ug/ml for the medium/low copy plasmids and 12.5 for the three control strains transformed with the R6K plasmid.<br />
<br />
<br />
'''Integration protocol:'''<br />
<br />
# Transform the <partinfo>BBa_J72008</partinfo> helper plasmid in the host strain (MC1061 or MG1655) and select transformants on Amp (50 ug/ml) plates under permissive conditions (30°C) overnight.<br />
# Inoculate a single colony in selective LB and let the culture grow at 30°C, 220 rpm. When the culture reaches the OD600 of 0.4-0.6 prepare chemically competent cells.<br />
# Transform the integrative vector with the desired insert in the BBa_J72008-containing strain and select co-transformants on Cm (34 ug/ml) plates under permissive conditions (30°C) overnight. At this temperature <partinfo>BBa_J72008</partinfo> can be replicated and so the pir protein product can be expressed in the cells. The pir product enables the propagation of the integrative vector by replicating the R6K origin.<br />
# Inoculate a single colony in 5 ml of LB + Cm at 12.5 ug/ml and incubate the culture at 37°C, 220 rpm overnight. At this temperature the <partinfo>BBa_J72008</partinfo> helper cannot be replicated and the Phi80 integrase is expressed by the remaining copies of the helper. The bacteria that are able to grow in this selective medium should be correct integrants because the integrative vector cannot be replicated by the pir product anymore.<br />
# Streak the culture on a Cm plate (at 12.5 ug/ml) and incubate it at 43°C overnight to ensure the loss of the helper plasmid. The bacteria that form colonies should be correct integrants without the <partinfo>BBa_J72008</partinfo> helper plasmid.<br />
<br />
Validate the loss of the helper plasmid by inoculating colonies in Cm (at 12.5 ug/ml) media and counterselecting them in Amp (at 50 ug/ml) media. Validate the correct integration position by performing colony PCR with primers P1/P2, P3/P4, P1/P4, P2,P3 and VF2/VR. Validate the phenotype (when possible).<br />
<br />
<br />
Expected amplicon length [bp] when the vector is integrated into the Phi80 locus:<br />
{|border=1<br />
|&nbsp;<br />
|'''No integrant'''<br />
|'''Single integrant'''<br />
|'''Multiple tandem integrants (>1)'''<br />
|-<br />
|'''VF2/VR'''<br />
|none<br />
|280 + insert length<br />
|280 + insert length<br />
|-<br />
|'''P1/P4'''<br />
|546<br />
|546 + insert length + 2171 (i.e. the BBa_K300000 length)<br />
|546 + insert length + 2171 (i.e. the BBa_K300000 length)<br />
|-<br />
|'''P1/P2'''<br />
|none<br />
|452<br />
|452<br />
|-<br />
|'''P3/P4'''<br />
|none<br />
|666<br />
|666<br />
|-<br />
|'''P2/P3'''<br />
|none<br />
|none<br />
|572<br />
|}<br />
<br />
<br />
'''Marker excision protocol:'''<br />
<br />
# Inoculate an integrant in selective LB medium and let it grow to OD600=0.4-0.6. Prepare chemically competent cells.<br />
# Transform the pCP20 helper plasmid in the competent strain and select transformants on Amp (100 ug/ml) plates under permissive conditions (30°C) overnight. At this temperature the pCP20 can be replicated. The pCP20 plasmid contains Amp and Cm resistance markers, a thermoinducible Flp recombinase expression system and a heat-sensitive replication origin. The permissive temperatures for the pCP20 propagation are the same as <partinfo>BBa_J72008</partinfo>.<br />
# Inoculate a single colony in 5 ml of LB without antibiotic and incubate the culture at 37°C, 220 rpm overnight. At this temperature the pCP20 helper cannot be replicated and the Flp recombinase is expressed by the remaining copies of the helper. The bacteria should loose the R6K origin and the Cm resistance upon FRT sites recombination, mediated by Flp.<br />
# Streak the culture on a LB plate and incubate it at 43°C overnight to ensure the loss of the helper plasmid. The bacteria that form colonies should be without the pCP20 helper plasmid.<br />
Validate the loss of the helper plasmid by inoculating colonies in Amp (at 100 ug/ml) media and validate the loss of the Cm resistance from the genome by inoculating colonies in Cm (at 12.5 ug/ml) media. Validate the correct length of the integrated part without Cm resistance and R6K origin by performing colony PCR with primers P1/P4 (which amplify the entire Phi80 locus) and VF2/VR (which amplify the integrated part). Validate the phenotype (when possible).<br />
<br />
<br />
'''Colony PCR:''' a single colony or 1 ul of culture was added to the Invitrogen Platinum Taq reaction mix and was heated at 94°C for 10 min. Then it was assayed with this cycle (X 35): 94°C 30 sec, 60°C (for VF2/VR) or 63°C (for the other primers) 30 sec, 72°C according to the amplicon expected length (1Kb/min). Then the reaction was kept at 72°C for 10 min and it was run on a 1% agarose gel with the GeneRuler 1Kb Plus DNA ladder (Fermentas).<br />
<br />
<br />
'''Fluorescence assays:''' integrants were inoculated in 1 ml of M9 + Cm (12.5 ug/ml) and grown at 37°C, 220 rpm overnight. The cultures were diluted 1:100 in 2 ml of selective M9 and let grow for about 4-6 hours under the same conditions as before. Three 200 ul aliquots for each culture were transferred to a 96-well microplate and assayed in the Infinite F200 microplate reader (Tecan) for about 20 hours with the following kinetic cycle: 37°C, 5 min sampling time, linear shaking 15 sec (amplitude=3), wait 5 sec, measure absorbance at 600nm, measure fluorescence with the proper filter (EX:nm/EM:540nm for GFP or EX:535nm/EM:620nm for RFP) with gain=70. The same protocol was followed for the MC1061 and the MG1655 non-integrant strains, which were grown in M9 without antibiotic.<br />
<br />
<br />
'''Data analysis:''' the absorbance measurements were normalized by subtracting the absorbance of the M9, while the fluorescence measurements were normalized by subtracting the fluorescence of the non-integrant strains over time. For each well, the S<sub>cell</sub> signal (proportional to the reporter protein synthesis rate per cell) was computed as (1/OD600*dXFP/dt), where OD600 is the normalized absorbance and XFP is the normalized fluorescence. The S<sub>cell</sub> signal was then averaged over time to obtain a single value for each well. Results are presented as the average S<sub>cell</sub> with their 95% confidence intervals of the mean.<br />
<br />
==Results==<br />
<br />
===Integration of the desired BioBrick part into the Phi80 genome locus===<br />
<br />
MC1061 and MG1655 were chosen as host strains for integration. <partinfo>BBa_K173001</partinfo> (constitutive strong promoter with GFPmut3) and the EcoRI-PstI fragment of <partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23101</partinfo> (here called PconRFP - constitutive strong promoter with RFP) were chosen as two proof of concept BioBrick parts to test the integration capability of the <partinfo>BBa_K300000</partinfo> vector in the Phi80 genome locus of these strains. For this reason, <partinfo>BBa_K173001</partinfo> and PconRFP were ligated in <partinfo>BBa_K300000</partinfo> (digested with EcoRI-PstI) and propagated using BW23474.<br />
The integration protocol was performed as described in the Materials and Methods section for 4 different combination:<br />
<br />
{|border=1<br />
|'''Integrant name'''<br />
|'''Strain'''<br />
|'''Insert of <partinfo>BBa_K300000</partinfo><br />
|-<br />
|MC-GFP<br />
|MC1061<br />
|<partinfo>BBa_K173001</partinfo><br />
|-<br />
|MC-RFP<br />
|MC1061<br />
|PconRFP<br />
|-<br />
|MG-GFP<br />
|MG1655<br />
|<partinfo>BBa_K173001</partinfo><br />
|-<br />
|MG-RFP<br />
|MG1655<br />
|PconRFP<br />
|}<br />
<br />
Three colonies grown after the overnight incubation at 43°C (step 5 of integration protocol) were analyzed for each plate. These 12 clones were called: MC-GFP-A,B,C , MC-RFP-A,B,C , MG-GFP-A,B,C and MG-RFP-A,B,C.<br />
<br />
<br />
'''Validation of the loss of BBa_J72008:''' all the picked colonies did not grow in Amp (50 ug/ml) media, thus validating that <partinfo>BBa_J72008</partinfo> Amp-resistant helper had been actually cured from the cells. However, one of these 12 clones (MG-GFP-A) also failed to grow in Cm (12.5 ug/ml) liquid media, probably because of a mistake in its inoculation. We decided not to consider this clone and to continue with 11 clones.<br />
<br />
<br />
'''Validation of the actual integration site:''' colony PCR was performed for all the 11 clones, using MC1061 and MG1655 without integrants as negative controls. Primer pairs P1/P2 and P3/P4 were used to validate the presence of the integrative vector in the Phi80 genomic locus, while the primer pair P2/P3 was used to validate the presence of multiple tandem integrants (see Fig.1 in Materials and Methods).<br />
<br />
<br />
{|align=center<br />
|[[Image:pv_P1P2.png|thumb|450px|center|Figure 2: colony PCR with P1/P2 on all the 11 integrant clones. The blank is the reaction mix without bacteria. Expected amplicon for correct integrants: 452 bp.]]<br />
|}<br />
<br />
{|align=center<br />
|[[Image:pv_P3P4.png|thumb|450px|center|Figure 3: colony PCR with P3/P4 on all the 11 integrant clones. The blank is the reaction mix without bacteria. Expected amplicon for correct integrants: 666 bp.]]<br />
|}<br />
<br />
{|align=center<br />
|[[Image:pv_P2P3.png|thumb|450px|center|Figure 4: colony PCR with P2/P3 on all the 11 integrant clones. The blank is the reaction mix without bacteria. The lanes with the amplicon were expected to come from bacteria with multiple tandem integrants. Expected amplicon for multiple integrants: 572 bp.]]<br />
|}<br />
<br />
<br />
PCR results with primers P1/P2 and P3/P4 showed that each clone had the correct integrant in the correct genomic position (see Materials and Methods for a list of the expected amplicon lengths). Negative controls showed no amplicons with primers P1/P2 as expected, but showed an unexpected band with P3/P4. The reason of the presence of this band was not further investigated and the results with this primer pair cannot be a useful tool for future analysis. Anyway, the P1/P2 primer pair can be sufficient to successfully validate the presence of the DNA of interest in the Phi80 genomic locus.<br />
<br />
PCR results with primers P2/P3 showed that two clones (MC-GFP-B and MC-GFP-C) were single integrants, while all the other clones were multiple tandem integrants (i.e. the Phi80 locus contained more than one copy of the DNA of interest). Negative controls showed no amplicons, as expected.<br />
<br />
<br />
'''Validation of the integrants phenotype:''' all the 11 clones were assayed as described in the Materials and Methods section. Unfortunately, the green fluorescent clones (MC-GFP-A,B,C and MG-GFP-B,C) did not show appreciable differences when compared to negative controls, most probably because the autofluorescence of the cells was too high and hid the GFP signal. For this reason, GFP clones were not considered for further analysis. Other instruments should be used to detect the GFP signal.<br />
<br />
On the other hand, RFP clones (MC-RFP-A,B,C and MG-RFP-A,B,C) all showed a higher fluorescence than the negative controls (see Fig.5). As Fig.5 show, the fluorescence of the three MG-RFP had a higher variability between clones when compared to the three MC-RFP. However, the clones were not necessarily expected to behave in the same way because all of them were multiple tandem integrants and the copy number of the PconRFP construct could be arbitrary.<br />
<br />
<br />
{|align=center<br />
|[[Image:pv_phenotypeRFPbefore.png|thumb|450px|center|Figure 5: relative RFP synthesis rate for all the RFP expressing clones. Note: as a reference, typical values of the relative RFP synthesis rate measured for PconRFP in a low copy vector (~5 plasmids per cell) are about 6-7 fold higher (data not shown).]]<br />
|}<br />
<br />
<br />
===Chloramphenicol resistance marker excision===<br />
<br />
The marker excision was performed on two of the previously validated integrant strains: MC-RFP-A and MG-RFP-A (even if they were multiple tandem integrants).<br />
<br />
The marker excision protocol was performed as described in the Materials and Methods section for both strains, here named:<br />
<br />
{|border=1<br />
|'''Original name'''<br />
|'''Name after marker excision'''<br />
|-<br />
|MC-RFP<br />
|MC-RFPflip<br />
|-<br />
|MG-RFP<br />
|MG-RFPflip<br />
|}<br />
<br />
<br />
Three colonies grown after the overnight incubation at 43°C (step 4 of marker excision protocol) were analyzed for each plate. These 6 clones were called MC-RFPflip-A,B,C and MG-FRPflip-A,B,C.<br />
<br />
<br />
'''Validation of the loss of pCP20 and the resistance marker:''' all the 6 picked colonies failed to grow on both Amp (100 ug/ml) media and Cm (12.5 ug/ml) media. They could only grow in LB without antibiotics, thus validating that the pCP20 helper had been actually cured and the R6K-CmR DNA containing the Chloramphenicol selection marker had been actually eliminated.<br />
<br />
<br />
'''Validation of the length of the integrated part:''' colony PCR was performed for all the 6 clones, using MC1061 and MG1655 without integrants as negative controls. Primer pairs VF2/VR and P1/P4 were used to validate if the ''passenger'' of interest was still present in the genome and the length of the entire Phi80 locus respectively after the marker excision.<br />
<br />
{|align=center<br />
|[[Image:pv_VF2VRintegrants.png|thumb|450px|center|Figure 6: colony PCR with VF2/VR on all the 6 flipped clones. The blank is the reaction mix without bacteria. Expected amplicon for correct insert: 1.2 Kb.]]<br />
|}<br />
<br />
{|align=center<br />
|[[Image:pv_P1P4integrants.png|thumb|450px|center|Figure 7: colony PCR with P1/P4 on all the 6 flipped clones. The blank is the reaction mix without bacteria. Expected amplicon for correct construct in the correct position: 2.3 Kb. Expected amplicon for the non-integrant strain MG1655: 546 bp.]]<br />
|}<br />
<br />
<br />
PCR results with primers VF2/VR showed that all the 6 clones still contain the ''passenger'' of interest, i.e. PconRFP, in the genome after the marker excision. The reaction blank, the MG1655 strain (neg control) and also the other samples showed some extra bands, but the ~1.2Kb amplicons of MC-RFP-A,B,C and MG-RFP-A,B,C had the correct length and was much brighter than the other bands.<br />
<br />
<br />
PCR results with primers P1/P4 (Fig.7) showed that an amplicon of ~2.3Kb was present in all but one screened clones, while the MG1655 negative control showed the expected 546bp length for a non-integrant. MC-RFPflip-C did not show the P1-P4 amplicon because the reaction failed: the tube was damaged and the reaction mix was completely evaporated at the end of the PCR program. For this reason, a PCR was performed again on this clone (Fig.8).<br />
<br />
The ~2.3Kb amplicon was consistent with a single integrant of <partinfo>BBa_K300000</partinfo>-PconRFP without the R6K-CmR DNA fragment, thus validating the successful excision of the FRT-flanked DNA fragment containing R6K-CmR and confirming that PconRFP was still present in the correct locus in single copy.<br />
<br />
<br />
{|align=center<br />
|[[Image:pv_P1P4singlecloneintegrant.png|thumb|300px|center|Figure 8: colony PCR with P1/P4 on MC-RFPflip-C clone. The blank is the reaction mix without bacteria. Expected amplicon for correct construct in the correct position: 2.3 Kb.]]<br />
|}<br />
<br />
<br />
These results showed that, even if the clones were multiple tandem integrants, they became single integrants after marker excision. This is because the Flp recombinase mediated the recombination of all the FRT sites of the multiple integrants until only a single FRT site was present in the Phi80 locus, thus leaving only the single integrant of interest without the selection marker in the genome.<br />
<br />
<br />
'''Validation of the marker-less phenotype:'''all the 6 clones were assayed as described in the Materials and Methods section. They all showed a low variability and their fluorescence was lower than their two ''parents'', i.e. MC-RFP-A for the MC1061 strains and MG-RFP-A for the MG1655 strains (see Fig.9). This result is consistent with the copy number of the PconRFP construct in the clones, in fact both MC-RFP-A and MG-RFP-A were multiple tandem integrants, while MC-RFPflip-A,B,C and MG-RFPflip-A,B,C were single integrants, as described above.<br />
<br />
All the MG-RFPflip showed a very low relative RFP synthesis rate when compared to the other strains, but the signal is systematically grater than the fluorescence of the negative control, thus validating the phenotype for the MG1655 strain. MC-RFPflip-A,B,C showed a higher fluorescence than MG-RFPflip-A,B,C.<br />
<br />
In conclusion, it has been demonstrated that, even after the marker excision process, the phenotype of the engineered cells is maintained.<br />
<br />
{|align=center<br />
|[[Image:pv_phenotypeRFP.png|thumb|450px|center|Figure 9: relative RFP synthesis rate for all the RFP-expressing clones after marker excision. In this figure, the bars corresponding to the fluorescence of the clones before marker excision is also reported to facilitate the comparison between them. Note that all the three ''flip'' clones are derived from MC-RFP-A for the MC1061 clones and from MG-RFP-A for the MG1655 clones.]]<br />
|}<br />
<br />
==Discussion==<br />
<br />
A novel integrative vector for ''E. coli'' has been successfully designed, constructed and used to integrate two proof of concept protein expression systems in two commonly used E. coli strains.<br />
<br />
The results showed that the vector is fully functional and can integrate into the correct targeted locus of the host chromosome through the Phi80 site-specific recombination system by using <partinfo>BBa_J72008</partinfo>, an existing BioBrick helper plasmid from the Registry. In most cases, the integration occurs in tandem copies, probably because of the too high Chloramphenicol concentration used during the selection of integrants, which forces multiple integration of Cm-resistant constructs. This concentration was the same used during the pSC101 low copy plasmid (~5 copies per cell) selection. In some cases, it is desirable to have a single copy of the desired BioBrick in the genome, for example when the gene dosage is important. In [Haldimann A and Wanner BL, 2001] the usage of Chloramphenicol at 6 ug/ml yielded a very high percentage of single integrants. However, when tested in our lab, the MG1655 strain could survive on LB plates with Cm at 6 ug/ml and also at 8 ug/ml. For this reason a higher concentration of Cm was chosen for selection. Further studies should investigate the optimal antibiotic concentration to yield the highest single integrants percentage as possible.<br />
<br />
<br />
The Flp/FRT mediated excision of the R6K and, most importantly, of the Cm resistance marker also worked by using the pCP20 helper plasmid. The estimated efficiency of this process was 100%. In addition, multiple tandem integrants became single integrants after the marker excision. This is because the Flp recombinase mediated the recombination of all the FRT sites of the multiple integrants until only a single FRT site was present in the Phi80 locus. The marker excision is a powerful tool to engineer microbial strains for industrial protein manufacturing because the engineered organism should not carry unsafe antibiotic resistances that may be diffused in the environment.<br />
<br />
<br />
The fluorescence phenotype confirmed the correct integration into the ''E. coli'' chromosome. As expected, in general multiple integrants showed a higher fluorescence than the single integrants.<br />
<br />
<br />
The BioBrick compatibility and the vector modularity give the possibility to the scientific community to stably engineer novel biological functions in ''E. coli'' with a very easy and user friendly methodology. A user’s handbook about the vector usage is shared in the Registry, as well as the users experiences and the compatibility information.<br />
<br />
<br />
<br />
=<partinfo>BBa_K300001</partinfo> - BioBrick integrative base vector for ''S. cerevisiae''=<br />
The integration capability of this vector has been tested in S288C ''S. cerevisiae'' strain (<partinfo>BBa_K300979</partinfo>). Here is reported the followed protocol and the obtained results.<br />
<br />
<br />
'''Protocol:'''<br />
<br />
*S288C strain (Open Biosystems) was inoculated in 5 ml of YPD from a long term 15% glycerol stock and grown for 24h (30°C, 200rpm).<br />
*The culture was diluted 1:10 in 50 ml of pre-warmed YPD in a 250 ml flask and was grown for additional 4 hours under the same conditions as before.<br />
*Cells were pelleted (4000 rpm, 5 min) and resuspended in 25 ml of deionized water.<br />
*Cells were pelleted (4000 rpm, 5 min), the supernatant was discarded and the pellet was resuspended in 1 ml of deionized water and transferred into a 1.5 ml tube.<br />
*Cells were pelleted (4000 rpm, 30 sec), the supernatant was discarded and the pellet was resuspended in deionized water to a final volume of 1 ml (vortex mix vigorously).<br />
*Three 100 ul aliquots were transferred into 1.5 ml tubes, while the remaining 600 ul of cells were not used in this protocol.<br />
*The three tubes were centrifuged (4000 rpm, 30 sec) and the supernatant discarded.<br />
*Each of the three pellets were resuspended (vortex mix vigorously) in 360 ul of transformation mix (240 ul of PEG 3350 50% w/v, 36 ul of LiAc 1.0 M, boiled salmon sperm DNA, 34 ul of linearized plasmid DNA plus water). The salmon sperm DNA was boiled for 5 min and pre-chilled before adding it in the transformation mix. The plasmid DNA was previously digested with SbfI (Fermentas), purified with the NucleoSpin Extract II kit (MN) and quantified with the NanoDrop in order to add 1 ug of DNA to the transformation mix.<br />
*The tubes were heated at 42°C for 40 min.<br />
*Cells were pelleted (4000 rpm, 30 sec), the supernatant was removed by pipetting and the pellet was gently resuspended in 1 ml of deionized water.<br />
*Cells were pelleted (4000 rpm, 30 sec), the supernatant was discarded, the pellet was resuspended in 1 ml of YPD and incubated at 30°C, 200 rpm for 3 hours.<br />
*Cells were pelleted (4000 rpm, 30 sec), resuspended in 200 ul of YPD and plated on a YPD agar plate with G418 antibiotic at 200 ug/ml.<br />
*The plates were incubated at 30°C for about 3 days until colonies appeared.<br />
<br />
<br />
The integration efficiency was estimated as the colony forming units (CFUs) yielded for each ug of DNA.<br />
<br />
<br />
Protocol references:<br />
<br />
[1] http://openwetware.org/wiki/High_Efficiency_Transformation<br />
<br />
[2] Guldener U, Heck S, Fiedler T, Beinhauer J, Hegemann JH (1996), A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Research, Vol. 24, No. 13 2519–2524.<br />
<br />
<br />
'''Results:'''<br />
<br />
The transformed inserts and their integration efficiency in S288C are listed here:<br />
<br />
{|border=1<br />
|'''SbfI-digested plasmid'''<br />
|'''ug of transformed DNA'''<br />
|'''# of colonies'''<br />
|'''Estimated integration efficiency [CFU/ug]'''<br />
|-<br />
|<partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300006</partinfo><br />
|1<br />
|1700<br />
|1.7*10^3<br />
|-<br />
|<partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300007</partinfo><br />
|1<br />
|6500<br />
|6.5*10^3<br />
|-<br />
|no DNA<br />
|0<br />
|0<br />
|0<br />
|}<br />
<br />
<br />
These results suggest that the integrative vector actually works and that the selection marker is highly specific (no colonies appeared on the "no DNA" plate).<br />
<br />
The correct phenotype of the S288C bearing these parts has still to be validated (by mOrange fluorescence measurement for the <partinfo>BBa_K300007</partinfo> part), as well as the actual integration position (by PCR).<br />
<br />
=<partinfo>BBa_K300004</partinfo> - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain=<br />
Inteins (Intervening Proteins) are sequences capable of self-exciding from a precursor protein through a process known as self-splicing, forming a peptide bond between the flanking proteins (exteins). Many so-called mini-Inteins have been engineered, whose key feature is the capability to completely release a flanking extein (the target protein) in response to a simple stimulus, either chemical or physical, with no need of expensive proteases.<br />
<br />
In literature, one mini-Intein was obtained through mutagenesis of ''Mycobacterium tuberculosis'' ''Mtu RecA'' Intein. The sequence of this Intein, referred to as ΔI-CM, allows for pH/heat-controlled C-terminal cleavage.<br />
<br />
Thanks to this feature, the ΔI-CM Intein can be fused downstream of an affinity tag and upstream of the protein coding sequence of interest in order to enable a cheap cleavage process to remove the N-terminal tag.<br />
<br />
The ΔI-CM Intein sequence was designed according to [Wood DW et al., 1999] and codon-optimized for ''E. coli'' to yield <partinfo>BBa_K300004</partinfo>. This part was designed as an internal domain (start/stop codons and it has Prefix and Suffix sequences compatible with RFC 23) in order to enable protein coding sequence assemblies to generate the desired synthetic self-cleavable affinity tags for protein purification.<br />
<br />
The optimal pH for cleavage has been reported to be 6.0, while a pH of 8.5 has been shown to inhibit the cleavage; C-terminus cleavage is also strongly affected by temperature >20°C [Wood DW et al., 1999]<br />
<br />
It was used to build together with phasins (<partinfo>BBa_K300002</partinfo> - head domain - and <partinfo>BBa_K300003</partinfo> - internal domain) and flexible protein domain linker (<partinfo>BBa_K105012</partinfo>) affinity tags that are able of self-cleaving to release the fusion protein of interest you need to purify:<br />
*<partinfo>BBa_K300095</partinfo><br />
*<partinfo>BBa_K300084</partinfo><br />
<br />
=<partinfo>BBa_K300010</partinfo> - PoPS-based self-inducible device=<br />
This is a PoPS-in/PoPS-out device.<br />
<br />
The luxR gene (<partinfo>BBa_C0062</partinfo>) is constitutively produced by the <partinfo>BBa_R0040</partinfo> promoter and it can activate the ''lux pR'' in presence of the autoinducer 3-oxo-C6-homoserine-lactone (3OC6HSL or simply HSL). The PoPS input regulates the production of luxI gene (<partinfo>BBa_C0061</partinfo>). It encodes for the LuxI enzyme, which is able to produce HSL. The produced HSL can diffuse in the growth media of the cells that express LuxI. The ''lux pR'' produces a PoPS output when HSL reaches a critical concentration.<br />
<br />
This device can be specialized by assembling a promoter upstream and a promoterless expression system with the gene of interest downstream. When a cell population expresses LuxI, the concentration of HSL is an increasing function of cell culture density and so the induction of the ''lux pR'' promoter occurs only when the cells reach a threshold density.<br />
<br />
In this way, the upstream promoter autoinduces the production of the target protein at a critical culture density, depending on the HSL synthesis rate. The HSL synthesis rate can be tuned by assembling promoters of different strengths upstream of ''luxI''.<br />
<br />
This enables the construction of a library of self-inducible devices capable of starting the target protein production at a predictable culture density.<br />
<br />
<br />
Source<br />
Receivers and Senders Registry parts.<br />
<br />
Design notes<br />
We used BioBrick Standard Assembly.<br />
<br />
<br />
This device has been characterized in many different experimental conditions:<br />
* varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation);<br />
* varying the copy number of vectors containing Sender and Receiver circuits;<br />
* varying the growth medium (LB or M9).<br />
<br />
The results obtained are reported in the sections below.<br />
<br />
=<partinfo>BBa_K300093</partinfo>, <partinfo>BBa_K300094</partinfo>, <partinfo>BBa_K300097</partinfo>, <partinfo>BBa_K300095</partinfo> and <partinfo>BBa_K300084</partinfo> - Phasin and Intein-based tags for protein purification=<br />
<br />
These parts are built by assembling Phasins (<partinfo>BBa_K300002</partinfo> and <partinfo>BBa_K300003</partinfo>) that are able to bind to PolyHydroxyAlkanoates (PHA)<br />
granules and Intein (<partinfo>BBa_K300004</partinfo>), a sequence capable of self-exciding from a precursor protein through a process known as self-splicing. In addition a flexible linker sequence (<partinfo>BBa_K105012</partinfo>) has been used to connect these parts in order to facilitate the binding and folding of the tag and the target protein of interest. Thanks to Phasin and Intein properties these parts can be used as high-specific TAGs for low-cost protein purification.<br />
<br />
At the moment were were not able to test Intein efficiency, but we could check if they affected the right folding of the target protein: we achieved this goal through the Silver Standard Assembly by using the GFP (<partinfo>BBa_K300005</partinfo>).<br />
<br />
These parts has been characterized respectively through:<br />
*pTet costitutive promoter devices:<br />
**<partinfo>BBa_K300088</partinfo><br />
**<partinfo>BBa_K300090</partinfo><br />
**<partinfo>BBa_K300099</partinfo><br />
*3OC6HSL inducible devices:<br />
**<partinfo>BBa_K300091</partinfo><br />
**<partinfo>BBa_K300092</partinfo><br />
and compared to a positive (<partinfo>BBa_K173000</partinfo>) and negative (<partinfo>BBa_B0031</partinfo>) control.<br />
<br />
==pTet costitutive promoter devices==<br />
===Methods===<br />
Inoculum (into 5 ml LB+Amp) from glycerol stock of:<br />
*<partinfo>BBa_K300088</partinfo><br />
*<partinfo>BBa_K300090</partinfo><br />
*<partinfo>BBa_K300099</partinfo><br />
*<partinfo>BBa_K173000</partinfo> (positive control)<br />
*<partinfo>BBa_B0031</partinfo> (negative control)<br />
They were let grow ON at +37°C, 220 rpm.<br />
<br />
The following day cultures were diluted 1:100 and let grow again for about five hours at +37°C, 220 rpm.<br />
<br />
Optical density (O.D.) of each cell culture was than measured with TECAN Infinte F200. Samples were diluted in order to obtain the same O.D. equal to 0,02.<br />
<br />
Than we performed a 21 hours' experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200. Each value shown is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted; cultures were shaken for 15 seconds every five minutes.<br />
<br />
===Results===<br />
<br />
{|align="center"<br />
|[[Image:UNIPV10_pTET_newP_ASB.png|thumb|300px|Raw growth curve]] || [[Image:UNIPV10_pTET_newP_GFP.png|thumb|300px|Raw GFP curve]]<br />
|}<br />
<table align="center"><br />
<tr><br />
<td><br />
[[Image:UNIPV10_pTET_newP_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<table border="1" align="center"><br />
<tr align="center"><br />
<th>Culture</th><th>Doubling time [min.] ± std error</th><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K173000</partinfo></td><td>76.3336 ± 1.4362</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300088</partinfo></td><td>74.8806 ± 2.7699</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300090</partinfo></td><td>75.9433 ± 3.6808</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300099</partinfo></td><td>78.4634 ± 2.5622</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_B0031</partinfo></td><td>70.8421 ± 2.2181</td><br />
</tr><br />
</table><br />
<br />
===Discussion===<br />
All cell cultures showed a similar growth curve and doubling time was computed as described [[Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation|here]] in order to have informations about the burden due to synthesis of such fusion proteins. It's possible to see that all doubling time are very similar; it's possible to assert that the expression of these BioBrick parts doesn't cause abnormal stress to the cells.<br />
<br />
In GFP curve it's possible to appreciate that in <partinfo>BBa_K300088</partinfo>, <partinfo>BBa_K300090</partinfo>, <partinfo>BBa_K300099</partinfo> GFP accumulation it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. These results show the right folding of the green fluorescent protein assembled downstream of the genetic circuit.<br />
<br />
The mean protein synthesis rate was also computed over the growth exponential phase, showing again an appreciable GFP production rate that is about a half of the positive control.<br />
<br />
==3OC6HSL inducible devices==<br />
===Methods===<br />
Inoculum (into 5 ml LB+Amp) from glycerol stock of:<br />
*<partinfo>BBa_K300091</partinfo><br />
*<partinfo>BBa_K300092</partinfo><br />
*<partinfo>BBa_K173000</partinfo> (positive control)<br />
*<partinfo>BBa_B0031</partinfo> (negative control)<br />
They were let grow ON at +37°C, 220 rpm.<br />
<br />
The following day cultures were diluted 1:100 and let grow again for about five hours at +37°C, 220 rpm.<br />
<br />
Optical density (O.D.) of each cell culture was than measured with TECAN Infinte F200. Samples were diluted in order to obtain the same O.D. equal to 0,02.<br />
<br />
Than we performed a 21 hours' experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200. <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> circuits were induced 100nM with HSL directly into multiplate well. Each value shown is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted; cultures were shaken for 15 seconds every five minutes.<br />
<br />
===Results===<br />
<table align="center"><br />
<tr><br />
<td>[[Image:UNIPV10_HSL_c_ASB.png|thumb|300px|Raw growth curve]]</td><br />
<td>[[Image:UNIPV10_HSL_c_GFP.png|thumb|300px|Raw GFP curve]]</td><br />
</tr><br />
</table><br />
<table align="center"><br />
<tr><br />
<td>[[Image:UNIPV10_HSL_c_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]</td><br />
</tr><br />
</table><br />
<br />
<table border="1" align="center"><br />
<tr align="center"><br />
<th>Culture</th><th>Doubling time [min.] ± std error</th><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K173000</partinfo></td><td>76.3336 ± 1.4362</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300091</partinfo><br/>induced</td><td>121.1434 ± 7.0275</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300091</partinfo><br/>not induced</td><td>74.4267 ± 1.3696</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300092</partinfo><br/>induced</td><td>122.6088 ± 1.2785</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300092</partinfo><br/>not induced</td><td>71.5105 ± 2.7113</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_B0031</partinfo></td><td>70.8421 ± 2.2181</td><br />
</tr><br />
</table><br />
<br />
===Discussion===<br />
All cell cultures showed a similar growth curve and doubling time was computed as described [[Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation|here]] in order to have informations about the burden due to synthesis of such fusion proteins. It's possible to see that all doubling time are very similar except for induced cultures. In this case doubling time is much higher than posite control and not induced cultures; so it's possible to assert that in this case there's a kind of metabolic burden higher than in the others, maybe because of the inducible system.<br />
<br />
In GFP curve it's possible to appreciate that in induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> GFP accumulation profile it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. On the other hand not induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> show a profile very similar to the last one. These results show the right folding of the green fluorescent protein assembled downstream of the genetic circuit and that the inducible system works as expected.<br />
<br />
The mean protein synthesis rate was also computed over the growth exponential phase, showing again an GFP production rate that is different from negative control. Not induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> show a low GFP synthesis rate maybe due to 3OC6HSL inducible circuit leakage activity.<br />
<br />
</td><br />
</tr></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewPartsTeam:UNIPV-Pavia/Parts/Characterization/NewParts2010-10-27T18:26:18Z<p>Susanna: /* BBa_K300010 - PoPS-based self-inducible device */</p>
<hr />
<div>__NOTOC__<br />
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<html><p align="center"><font size="4"><b>NEW PARTS</b></font></p></html><hr><br />
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[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization]<br />
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[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
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[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
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[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
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</table><br><br />
<br />
=New Parts: list=<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300000 - BioBrick integrative base vector for E. coli |BBa_K300000 - BioBrick integrative base vector for E. coli]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300001 - BioBrick integrative base vector for S. cerevisiae|BBa_K300001 - BioBrick integrative base vector for S. cerevisiae]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain|BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300010 - PoPS-based self-inducible device|BBa_K300010 - PoPS-based self-inducible device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification|BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification]]<br />
<br />
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----<br />
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<tr><td><br />
<br />
=<partinfo>BBa_K300000</partinfo> - BioBrick integrative base vector for ''E. coli''=<br />
<br />
==Materials and Methods==<br />
<br />
'''Plasmids and strains:''' the <partinfo>BBa_J72008</partinfo> helper plasmid was kindly given by Prof. JC Anderson (UC Berkeley). BW23474 (<partinfo>BBa_K300985</partinfo>), MC1061 (<partinfo>BBa_K300078</partinfo>) and MG1655 (<partinfo>BBa_V1000</partinfo>) E. coli strains and the pCP20 helper plasmid were purchased from the Coli Genetic Stock Center (Yale University).<br />
<br />
<br />
'''Verification primers:''' all the oligonucleotides were purchased from Primm (San Raffaele Biomedical Science Park, Milan, Italy). The P1 (<partinfo>BBa_K300975</partinfo>) and P4 (<partinfo>BBa_K300978</partinfo>) primers had already been used in [Anderson JC et al., 2010]. The P2 (<partinfo>BBa_K300976</partinfo>) and P3 (<partinfo>BBa_K300977</partinfo>) primers have been newly designed using ApE and Amplify 3X. P2 and P3 have been designed also considering the previously used verification primers P2 and P3 in the pG80ko integrative plasmid, described in [DeLoache W, 2009].<br />
<br />
The relative position of the P1, P2, P3 and P4 primers is shown in Fig.1:<br />
<br />
{|align=center<br />
|[[Image:relativeprimers.png|thumb|450px|center|Figure 1: Relative position of the verification primers. a) no integrants; b) single integrant and c) integrant with multiple tandem copies. P1/P2 and P3/P4 pairs give an amplicon when at least one copy of the vector is integrated in the Phi80 locus. P2/P3 pair show an amplicon only when multiple tandem copies occur.]]<br />
|}<br />
<br />
'''Competent cells preparation:''' all the ''E. coli'' strains were made competent following a slightly modified version of the protocol described in [Sambrook J et al., 1989]. Briefly, cells were grown to and OD600 of ~0.4-0.6, harvested (4000 rpm, 10 min, 4°C) and the supernatant discarded. Cells were resuspended in (30 ml for each 50 ml of initial culture) pre-chilled Mg-Ca buffer (80 mM MgCl2, 20 mM CaCl2), centrifuged as before and the supernatant discarded. Cells were resuspended in (2 ml for each 50 ml of initial culture) pre-chilled Ca buffer (100 mM CaCl2, 15% glycerol), aliquoted in 0.5 ml tubes and freezed immediately at -80°C. Test the transformation efficiency in Colony Forming Units (CFU)/ug of transformed DNA<br />
<br />
The Chloramphenicol concentration in plates was 34 ug/ml for the high copy plasmids, 12.5 ug/ml for the medium/low copy plasmids and 12.5 for the three control strains transformed with the R6K plasmid.<br />
<br />
<br />
'''Integration protocol:'''<br />
<br />
# Transform the <partinfo>BBa_J72008</partinfo> helper plasmid in the host strain (MC1061 or MG1655) and select transformants on Amp (50 ug/ml) plates under permissive conditions (30°C) overnight.<br />
# Inoculate a single colony in selective LB and let the culture grow at 30°C, 220 rpm. When the culture reaches the OD600 of 0.4-0.6 prepare chemically competent cells.<br />
# Transform the integrative vector with the desired insert in the BBa_J72008-containing strain and select co-transformants on Cm (34 ug/ml) plates under permissive conditions (30°C) overnight. At this temperature <partinfo>BBa_J72008</partinfo> can be replicated and so the pir protein product can be expressed in the cells. The pir product enables the propagation of the integrative vector by replicating the R6K origin.<br />
# Inoculate a single colony in 5 ml of LB + Cm at 12.5 ug/ml and incubate the culture at 37°C, 220 rpm overnight. At this temperature the <partinfo>BBa_J72008</partinfo> helper cannot be replicated and the Phi80 integrase is expressed by the remaining copies of the helper. The bacteria that are able to grow in this selective medium should be correct integrants because the integrative vector cannot be replicated by the pir product anymore.<br />
# Streak the culture on a Cm plate (at 12.5 ug/ml) and incubate it at 43°C overnight to ensure the loss of the helper plasmid. The bacteria that form colonies should be correct integrants without the <partinfo>BBa_J72008</partinfo> helper plasmid.<br />
<br />
Validate the loss of the helper plasmid by inoculating colonies in Cm (at 12.5 ug/ml) media and counterselecting them in Amp (at 50 ug/ml) media. Validate the correct integration position by performing colony PCR with primers P1/P2, P3/P4, P1/P4, P2,P3 and VF2/VR. Validate the phenotype (when possible).<br />
<br />
<br />
Expected amplicon length [bp] when the vector is integrated into the Phi80 locus:<br />
{|border=1<br />
|&nbsp;<br />
|'''No integrant'''<br />
|'''Single integrant'''<br />
|'''Multiple tandem integrants (>1)'''<br />
|-<br />
|'''VF2/VR'''<br />
|none<br />
|280 + insert length<br />
|280 + insert length<br />
|-<br />
|'''P1/P4'''<br />
|546<br />
|546 + insert length + 2171 (i.e. the BBa_K300000 length)<br />
|546 + insert length + 2171 (i.e. the BBa_K300000 length)<br />
|-<br />
|'''P1/P2'''<br />
|none<br />
|452<br />
|452<br />
|-<br />
|'''P3/P4'''<br />
|none<br />
|666<br />
|666<br />
|-<br />
|'''P2/P3'''<br />
|none<br />
|none<br />
|572<br />
|}<br />
<br />
<br />
'''Marker excision protocol:'''<br />
<br />
# Inoculate an integrant in selective LB medium and let it grow to OD600=0.4-0.6. Prepare chemically competent cells.<br />
# Transform the pCP20 helper plasmid in the competent strain and select transformants on Amp (100 ug/ml) plates under permissive conditions (30°C) overnight. At this temperature the pCP20 can be replicated. The pCP20 plasmid contains Amp and Cm resistance markers, a thermoinducible Flp recombinase expression system and a heat-sensitive replication origin. The permissive temperatures for the pCP20 propagation are the same as <partinfo>BBa_J72008</partinfo>.<br />
# Inoculate a single colony in 5 ml of LB without antibiotic and incubate the culture at 37°C, 220 rpm overnight. At this temperature the pCP20 helper cannot be replicated and the Flp recombinase is expressed by the remaining copies of the helper. The bacteria should loose the R6K origin and the Cm resistance upon FRT sites recombination, mediated by Flp.<br />
# Streak the culture on a LB plate and incubate it at 43°C overnight to ensure the loss of the helper plasmid. The bacteria that form colonies should be without the pCP20 helper plasmid.<br />
Validate the loss of the helper plasmid by inoculating colonies in Amp (at 100 ug/ml) media and validate the loss of the Cm resistance from the genome by inoculating colonies in Cm (at 12.5 ug/ml) media. Validate the correct length of the integrated part without Cm resistance and R6K origin by performing colony PCR with primers P1/P4 (which amplify the entire Phi80 locus) and VF2/VR (which amplify the integrated part). Validate the phenotype (when possible).<br />
<br />
<br />
'''Colony PCR:''' a single colony or 1 ul of culture was added to the Invitrogen Platinum Taq reaction mix and was heated at 94°C for 10 min. Then it was assayed with this cycle (X 35): 94°C 30 sec, 60°C (for VF2/VR) or 63°C (for the other primers) 30 sec, 72°C according to the amplicon expected length (1Kb/min). Then the reaction was kept at 72°C for 10 min and it was run on a 1% agarose gel with the GeneRuler 1Kb Plus DNA ladder (Fermentas).<br />
<br />
<br />
'''Fluorescence assays:''' integrants were inoculated in 1 ml of M9 + Cm (12.5 ug/ml) and grown at 37°C, 220 rpm overnight. The cultures were diluted 1:100 in 2 ml of selective M9 and let grow for about 4-6 hours under the same conditions as before. Three 200 ul aliquots for each culture were transferred to a 96-well microplate and assayed in the Infinite F200 microplate reader (Tecan) for about 20 hours with the following kinetic cycle: 37°C, 5 min sampling time, linear shaking 15 sec (amplitude=3), wait 5 sec, measure absorbance at 600nm, measure fluorescence with the proper filter (EX:nm/EM:540nm for GFP or EX:535nm/EM:620nm for RFP) with gain=70. The same protocol was followed for the MC1061 and the MG1655 non-integrant strains, which were grown in M9 without antibiotic.<br />
<br />
<br />
'''Data analysis:''' the absorbance measurements were normalized by subtracting the absorbance of the M9, while the fluorescence measurements were normalized by subtracting the fluorescence of the non-integrant strains over time. For each well, the S<sub>cell</sub> signal (proportional to the reporter protein synthesis rate per cell) was computed as (1/OD600*dXFP/dt), where OD600 is the normalized absorbance and XFP is the normalized fluorescence. The S<sub>cell</sub> signal was then averaged over time to obtain a single value for each well. Results are presented as the average S<sub>cell</sub> with their 95% confidence intervals of the mean.<br />
<br />
==Results==<br />
<br />
===Integration of the desired BioBrick part into the Phi80 genome locus===<br />
<br />
MC1061 and MG1655 were chosen as host strains for integration. <partinfo>BBa_K173001</partinfo> (constitutive strong promoter with GFPmut3) and the EcoRI-PstI fragment of <partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23101</partinfo> (here called PconRFP - constitutive strong promoter with RFP) were chosen as two proof of concept BioBrick parts to test the integration capability of the <partinfo>BBa_K300000</partinfo> vector in the Phi80 genome locus of these strains. For this reason, <partinfo>BBa_K173001</partinfo> and PconRFP were ligated in <partinfo>BBa_K300000</partinfo> (digested with EcoRI-PstI) and propagated using BW23474.<br />
The integration protocol was performed as described in the Materials and Methods section for 4 different combination:<br />
<br />
{|border=1<br />
|'''Integrant name'''<br />
|'''Strain'''<br />
|'''Insert of <partinfo>BBa_K300000</partinfo><br />
|-<br />
|MC-GFP<br />
|MC1061<br />
|<partinfo>BBa_K173001</partinfo><br />
|-<br />
|MC-RFP<br />
|MC1061<br />
|PconRFP<br />
|-<br />
|MG-GFP<br />
|MG1655<br />
|<partinfo>BBa_K173001</partinfo><br />
|-<br />
|MG-RFP<br />
|MG1655<br />
|PconRFP<br />
|}<br />
<br />
Three colonies grown after the overnight incubation at 43°C (step 5 of integration protocol) were analyzed for each plate. These 12 clones were called: MC-GFP-A,B,C , MC-RFP-A,B,C , MG-GFP-A,B,C and MG-RFP-A,B,C.<br />
<br />
<br />
'''Validation of the loss of BBa_J72008:''' all the picked colonies did not grow in Amp (50 ug/ml) media, thus validating that <partinfo>BBa_J72008</partinfo> Amp-resistant helper had been actually cured from the cells. However, one of these 12 clones (MG-GFP-A) also failed to grow in Cm (12.5 ug/ml) liquid media, probably because of a mistake in its inoculation. We decided not to consider this clone and to continue with 11 clones.<br />
<br />
<br />
'''Validation of the actual integration site:''' colony PCR was performed for all the 11 clones, using MC1061 and MG1655 without integrants as negative controls. Primer pairs P1/P2 and P3/P4 were used to validate the presence of the integrative vector in the Phi80 genomic locus, while the primer pair P2/P3 was used to validate the presence of multiple tandem integrants (see Fig.1 in Materials and Methods).<br />
<br />
<br />
{|align=center<br />
|[[Image:pv_P1P2.png|thumb|450px|center|Figure 2: colony PCR with P1/P2 on all the 11 integrant clones. The blank is the reaction mix without bacteria. Expected amplicon for correct integrants: 452 bp.]]<br />
|}<br />
<br />
{|align=center<br />
|[[Image:pv_P3P4.png|thumb|450px|center|Figure 3: colony PCR with P3/P4 on all the 11 integrant clones. The blank is the reaction mix without bacteria. Expected amplicon for correct integrants: 666 bp.]]<br />
|}<br />
<br />
{|align=center<br />
|[[Image:pv_P2P3.png|thumb|450px|center|Figure 4: colony PCR with P2/P3 on all the 11 integrant clones. The blank is the reaction mix without bacteria. The lanes with the amplicon were expected to come from bacteria with multiple tandem integrants. Expected amplicon for multiple integrants: 572 bp.]]<br />
|}<br />
<br />
<br />
PCR results with primers P1/P2 and P3/P4 showed that each clone had the correct integrant in the correct genomic position (see Materials and Methods for a list of the expected amplicon lengths). Negative controls showed no amplicons with primers P1/P2 as expected, but showed an unexpected band with P3/P4. The reason of the presence of this band was not further investigated and the results with this primer pair cannot be a useful tool for future analysis. Anyway, the P1/P2 primer pair can be sufficient to successfully validate the presence of the DNA of interest in the Phi80 genomic locus.<br />
<br />
PCR results with primers P2/P3 showed that two clones (MC-GFP-B and MC-GFP-C) were single integrants, while all the other clones were multiple tandem integrants (i.e. the Phi80 locus contained more than one copy of the DNA of interest). Negative controls showed no amplicons, as expected.<br />
<br />
<br />
'''Validation of the integrants phenotype:''' all the 11 clones were assayed as described in the Materials and Methods section. Unfortunately, the green fluorescent clones (MC-GFP-A,B,C and MG-GFP-B,C) did not show appreciable differences when compared to negative controls, most probably because the autofluorescence of the cells was too high and hid the GFP signal. For this reason, GFP clones were not considered for further analysis. Other instruments should be used to detect the GFP signal.<br />
<br />
On the other hand, RFP clones (MC-RFP-A,B,C and MG-RFP-A,B,C) all showed a higher fluorescence than the negative controls (see Fig.5). As Fig.5 show, the fluorescence of the three MG-RFP had a higher variability between clones when compared to the three MC-RFP. However, the clones were not necessarily expected to behave in the same way because all of them were multiple tandem integrants and the copy number of the PconRFP construct could be arbitrary.<br />
<br />
<br />
{|align=center<br />
|[[Image:pv_phenotypeRFPbefore.png|thumb|450px|center|Figure 5: relative RFP synthesis rate for all the RFP expressing clones. Note: as a reference, typical values of the relative RFP synthesis rate measured for PconRFP in a low copy vector (~5 plasmids per cell) are about 6-7 fold higher (data not shown).]]<br />
|}<br />
<br />
<br />
===Chloramphenicol resistance marker excision===<br />
<br />
The marker excision was performed on two of the previously validated integrant strains: MC-RFP-A and MG-RFP-A (even if they were multiple tandem integrants).<br />
<br />
The marker excision protocol was performed as described in the Materials and Methods section for both strains, here named:<br />
<br />
{|border=1<br />
|'''Original name'''<br />
|'''Name after marker excision'''<br />
|-<br />
|MC-RFP<br />
|MC-RFPflip<br />
|-<br />
|MG-RFP<br />
|MG-RFPflip<br />
|}<br />
<br />
<br />
Three colonies grown after the overnight incubation at 43°C (step 4 of marker excision protocol) were analyzed for each plate. These 6 clones were called MC-RFPflip-A,B,C and MG-FRPflip-A,B,C.<br />
<br />
<br />
'''Validation of the loss of pCP20 and the resistance marker:''' all the 6 picked colonies failed to grow on both Amp (100 ug/ml) media and Cm (12.5 ug/ml) media. They could only grow in LB without antibiotics, thus validating that the pCP20 helper had been actually cured and the R6K-CmR DNA containing the Chloramphenicol selection marker had been actually eliminated.<br />
<br />
<br />
'''Validation of the length of the integrated part:''' colony PCR was performed for all the 6 clones, using MC1061 and MG1655 without integrants as negative controls. Primer pairs VF2/VR and P1/P4 were used to validate if the ''passenger'' of interest was still present in the genome and the length of the entire Phi80 locus respectively after the marker excision.<br />
<br />
{|align=center<br />
|[[Image:pv_VF2VRintegrants.png|thumb|450px|center|Figure 6: colony PCR with VF2/VR on all the 6 flipped clones. The blank is the reaction mix without bacteria. Expected amplicon for correct insert: 1.2 Kb.]]<br />
|}<br />
<br />
{|align=center<br />
|[[Image:pv_P1P4integrants.png|thumb|450px|center|Figure 7: colony PCR with P1/P4 on all the 6 flipped clones. The blank is the reaction mix without bacteria. Expected amplicon for correct construct in the correct position: 2.3 Kb. Expected amplicon for the non-integrant strain MG1655: 546 bp.]]<br />
|}<br />
<br />
<br />
PCR results with primers VF2/VR showed that all the 6 clones still contain the ''passenger'' of interest, i.e. PconRFP, in the genome after the marker excision. The reaction blank, the MG1655 strain (neg control) and also the other samples showed some extra bands, but the ~1.2Kb amplicons of MC-RFP-A,B,C and MG-RFP-A,B,C had the correct length and was much brighter than the other bands.<br />
<br />
<br />
PCR results with primers P1/P4 (Fig.7) showed that an amplicon of ~2.3Kb was present in all but one screened clones, while the MG1655 negative control showed the expected 546bp length for a non-integrant. MC-RFPflip-C did not show the P1-P4 amplicon because the reaction failed: the tube was damaged and the reaction mix was completely evaporated at the end of the PCR program. For this reason, a PCR was performed again on this clone (Fig.8).<br />
<br />
The ~2.3Kb amplicon was consistent with a single integrant of <partinfo>BBa_K300000</partinfo>-PconRFP without the R6K-CmR DNA fragment, thus validating the successful excision of the FRT-flanked DNA fragment containing R6K-CmR and confirming that PconRFP was still present in the correct locus in single copy.<br />
<br />
<br />
{|align=center<br />
|[[Image:pv_P1P4singlecloneintegrant.png|thumb|300px|center|Figure 8: colony PCR with P1/P4 on MC-RFPflip-C clone. The blank is the reaction mix without bacteria. Expected amplicon for correct construct in the correct position: 2.3 Kb.]]<br />
|}<br />
<br />
<br />
These results showed that, even if the clones were multiple tandem integrants, they became single integrants after marker excision. This is because the Flp recombinase mediated the recombination of all the FRT sites of the multiple integrants until only a single FRT site was present in the Phi80 locus, thus leaving only the single integrant of interest without the selection marker in the genome.<br />
<br />
<br />
'''Validation of the marker-less phenotype:'''all the 6 clones were assayed as described in the Materials and Methods section. They all showed a low variability and their fluorescence was lower than their two ''parents'', i.e. MC-RFP-A for the MC1061 strains and MG-RFP-A for the MG1655 strains (see Fig.9). This result is consistent with the copy number of the PconRFP construct in the clones, in fact both MC-RFP-A and MG-RFP-A were multiple tandem integrants, while MC-RFPflip-A,B,C and MG-RFPflip-A,B,C were single integrants, as described above.<br />
<br />
All the MG-RFPflip showed a very low relative RFP synthesis rate when compared to the other strains, but the signal is systematically grater than the fluorescence of the negative control, thus validating the phenotype for the MG1655 strain. MC-RFPflip-A,B,C showed a higher fluorescence than MG-RFPflip-A,B,C.<br />
<br />
In conclusion, it has been demonstrated that, even after the marker excision process, the phenotype of the engineered cells is maintained.<br />
<br />
{|align=center<br />
|[[Image:pv_phenotypeRFP.png|thumb|450px|center|Figure 9: relative RFP synthesis rate for all the RFP-expressing clones after marker excision. In this figure, the bars corresponding to the fluorescence of the clones before marker excision is also reported to facilitate the comparison between them. Note that all the three ''flip'' clones are derived from MC-RFP-A for the MC1061 clones and from MG-RFP-A for the MG1655 clones.]]<br />
|}<br />
<br />
==Discussion==<br />
<br />
A novel integrative vector for ''E. coli'' has been successfully designed, constructed and used to integrate two proof of concept protein expression systems in two commonly used E. coli strains.<br />
<br />
The results showed that the vector is fully functional and can integrate into the correct targeted locus of the host chromosome through the Phi80 site-specific recombination system by using <partinfo>BBa_J72008</partinfo>, an existing BioBrick helper plasmid from the Registry. In most cases, the integration occurs in tandem copies, probably because of the too high Chloramphenicol concentration used during the selection of integrants, which forces multiple integration of Cm-resistant constructs. This concentration was the same used during the pSC101 low copy plasmid (~5 copies per cell) selection. In some cases, it is desirable to have a single copy of the desired BioBrick in the genome, for example when the gene dosage is important. In [Haldimann A and Wanner BL, 2001] the usage of Chloramphenicol at 6 ug/ml yielded a very high percentage of single integrants. However, when tested in our lab, the MG1655 strain could survive on LB plates with Cm at 6 ug/ml and also at 8 ug/ml. For this reason a higher concentration of Cm was chosen for selection. Further studies should investigate the optimal antibiotic concentration to yield the highest single integrants percentage as possible.<br />
<br />
<br />
The Flp/FRT mediated excision of the R6K and, most importantly, of the Cm resistance marker also worked by using the pCP20 helper plasmid. The estimated efficiency of this process was 100%. In addition, multiple tandem integrants became single integrants after the marker excision. This is because the Flp recombinase mediated the recombination of all the FRT sites of the multiple integrants until only a single FRT site was present in the Phi80 locus. The marker excision is a powerful tool to engineer microbial strains for industrial protein manufacturing because the engineered organism should not carry unsafe antibiotic resistances that may be diffused in the environment.<br />
<br />
<br />
The fluorescence phenotype confirmed the correct integration into the ''E. coli'' chromosome. As expected, in general multiple integrants showed a higher fluorescence than the single integrants.<br />
<br />
<br />
The BioBrick compatibility and the vector modularity give the possibility to the scientific community to stably engineer novel biological functions in ''E. coli'' with a very easy and user friendly methodology. A user’s handbook about the vector usage is shared in the Registry, as well as the users experiences and the compatibility information.<br />
<br />
<br />
<br />
=<partinfo>BBa_K300001</partinfo> - BioBrick integrative base vector for ''S. cerevisiae''=<br />
The integration capability of this vector has been tested in S288C ''S. cerevisiae'' strain (<partinfo>BBa_K300979</partinfo>). Here is reported the followed protocol and the obtained results.<br />
<br />
<br />
'''Protocol:'''<br />
<br />
*S288C strain (Open Biosystems) was inoculated in 5 ml of YPD from a long term 15% glycerol stock and grown for 24h (30°C, 200rpm).<br />
*The culture was diluted 1:10 in 50 ml of pre-warmed YPD in a 250 ml flask and was grown for additional 4 hours under the same conditions as before.<br />
*Cells were pelleted (4000 rpm, 5 min) and resuspended in 25 ml of deionized water.<br />
*Cells were pelleted (4000 rpm, 5 min), the supernatant was discarded and the pellet was resuspended in 1 ml of deionized water and transferred into a 1.5 ml tube.<br />
*Cells were pelleted (4000 rpm, 30 sec), the supernatant was discarded and the pellet was resuspended in deionized water to a final volume of 1 ml (vortex mix vigorously).<br />
*Three 100 ul aliquots were transferred into 1.5 ml tubes, while the remaining 600 ul of cells were not used in this protocol.<br />
*The three tubes were centrifuged (4000 rpm, 30 sec) and the supernatant discarded.<br />
*Each of the three pellets were resuspended (vortex mix vigorously) in 360 ul of transformation mix (240 ul of PEG 3350 50% w/v, 36 ul of LiAc 1.0 M, boiled salmon sperm DNA, 34 ul of linearized plasmid DNA plus water). The salmon sperm DNA was boiled for 5 min and pre-chilled before adding it in the transformation mix. The plasmid DNA was previously digested with SbfI (Fermentas), purified with the NucleoSpin Extract II kit (MN) and quantified with the NanoDrop in order to add 1 ug of DNA to the transformation mix.<br />
*The tubes were heated at 42°C for 40 min.<br />
*Cells were pelleted (4000 rpm, 30 sec), the supernatant was removed by pipetting and the pellet was gently resuspended in 1 ml of deionized water.<br />
*Cells were pelleted (4000 rpm, 30 sec), the supernatant was discarded, the pellet was resuspended in 1 ml of YPD and incubated at 30°C, 200 rpm for 3 hours.<br />
*Cells were pelleted (4000 rpm, 30 sec), resuspended in 200 ul of YPD and plated on a YPD agar plate with G418 antibiotic at 200 ug/ml.<br />
*The plates were incubated at 30°C for about 3 days until colonies appeared.<br />
<br />
<br />
The integration efficiency was estimated as the colony forming units (CFUs) yielded for each ug of DNA.<br />
<br />
<br />
Protocol references:<br />
<br />
[1] http://openwetware.org/wiki/High_Efficiency_Transformation<br />
<br />
[2] Guldener U, Heck S, Fiedler T, Beinhauer J, Hegemann JH (1996), A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Research, Vol. 24, No. 13 2519–2524.<br />
<br />
<br />
'''Results:'''<br />
<br />
The transformed inserts and their integration efficiency in S288C are listed here:<br />
<br />
{|border=1<br />
|'''SbfI-digested plasmid'''<br />
|'''ug of transformed DNA'''<br />
|'''# of colonies'''<br />
|'''Estimated integration efficiency [CFU/ug]'''<br />
|-<br />
|<partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300006</partinfo><br />
|1<br />
|1700<br />
|1.7*10^3<br />
|-<br />
|<partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300007</partinfo><br />
|1<br />
|6500<br />
|6.5*10^3<br />
|-<br />
|no DNA<br />
|0<br />
|0<br />
|0<br />
|}<br />
<br />
<br />
These results suggest that the integrative vector actually works and that the selection marker is highly specific (no colonies appeared on the "no DNA" plate).<br />
<br />
The correct phenotype of the S288C bearing these parts has still to be validated (by mOrange fluorescence measurement for the <partinfo>BBa_K300007</partinfo> part), as well as the actual integration position (by PCR).<br />
<br />
=<partinfo>BBa_K300004</partinfo> - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain=<br />
Inteins (Intervening Proteins) are sequences capable of self-exciding from a precursor protein through a process known as self-splicing, forming a peptide bond between the flanking proteins (exteins). Many so-called mini-Inteins have been engineered, whose key feature is the capability to completely release a flanking extein (the target protein) in response to a simple stimulus, either chemical or physical, with no need of expensive proteases.<br />
<br />
In literature, one mini-Intein was obtained through mutagenesis of ''Mycobacterium tuberculosis'' ''Mtu RecA'' Intein. The sequence of this Intein, referred to as ΔI-CM, allows for pH/heat-controlled C-terminal cleavage.<br />
<br />
Thanks to this feature, the ΔI-CM Intein can be fused downstream of an affinity tag and upstream of the protein coding sequence of interest in order to enable a cheap cleavage process to remove the N-terminal tag.<br />
<br />
The ΔI-CM Intein sequence was designed according to [Wood DW et al., 1999] and codon-optimized for ''E. coli'' to yield <partinfo>BBa_K300004</partinfo>. This part was designed as an internal domain (start/stop codons and it has Prefix and Suffix sequences compatible with RFC 23) in order to enable protein coding sequence assemblies to generate the desired synthetic self-cleavable affinity tags for protein purification.<br />
<br />
The optimal pH for cleavage has been reported to be 6.0, while a pH of 8.5 has been shown to inhibit the cleavage; C-terminus cleavage is also strongly affected by temperature >20°C [Wood DW et al., 1999]<br />
<br />
It was used to build together with phasins (<partinfo>BBa_K300002</partinfo> - head domain - and <partinfo>BBa_K300003</partinfo> - internal domain) and flexible protein domain linker (<partinfo>BBa_K105012</partinfo>) affinity tags that are able of self-cleaving to release the fusion protein of interest you need to purify:<br />
*<partinfo>BBa_K300095</partinfo><br />
*<partinfo>BBa_K300084</partinfo><br />
<br />
=<partinfo>BBa_K300010</partinfo> - PoPS-based self-inducible device=<br />
<br />
This is a PoPS-in/PoPS-out device.<br />
<br />
The luxR gene (<partinfo>BBa_C0062</partinfo>) is constitutively produced by the <partinfo>BBa_R0040</partinfo> promoter and it can activate the ''lux pR'' in presence of the autoinducer 3-oxo-C6-homoserine-lactone (3OC6HSL or simply HSL). The PoPS input regulates the production of luxI gene (<partinfo>BBa_C0061</partinfo>). It encodes for the LuxI enzyme, which is able to produce HSL. The produced HSL can diffuse in the growth media of the cells that express LuxI. The ''lux pR'' produces a PoPS output when HSL reaches a critical concentration.<br />
<br />
This device can be specialized by assembling a promoter upstream and a promoterless expression system with the gene of interest downstream. When a cell population expresses luxI, the concentration of HSL is an increasing function of cell culture density and so the induction of the lux pR promoter occurs only when the cells reach a threshold density.<br />
<br />
In this way, the upstream promoter autoinduces the production of the target protein at a critical culture density, depending on the HSL synthesis rate. The HSL synthesis rate can be tuned by assembling promoters of different strengths upstream of luxI.<br />
<br />
This enables the construction of a library of self-inducible devices capable of starting the target protein production at a predictable culture density.<br />
<br />
<br />
Source<br />
Receivers and Senders Registry parts.<br />
<br />
Design notes<br />
We used BioBrick Standard Assembly.<br />
<br />
<br />
This device has been characterized in many different experimental conditions:<br />
* varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation);<br />
* varying the copy number of vectors containing Sender and Receiver circuits;<br />
* varying the growth medium (LB or M9).<br />
<br />
The results obtained are reported in the sections below.<br />
<br />
=<partinfo>BBa_K300093</partinfo>, <partinfo>BBa_K300094</partinfo>, <partinfo>BBa_K300097</partinfo>, <partinfo>BBa_K300095</partinfo> and <partinfo>BBa_K300084</partinfo> - Phasin and Intein-based tags for protein purification=<br />
<br />
These parts are built by assembling Phasins (<partinfo>BBa_K300002</partinfo> and <partinfo>BBa_K300003</partinfo>) that are able to bind to PolyHydroxyAlkanoates (PHA)<br />
granules and Intein (<partinfo>BBa_K300004</partinfo>), a sequence capable of self-exciding from a precursor protein through a process known as self-splicing. In addition a flexible linker sequence (<partinfo>BBa_K105012</partinfo>) has been used to connect these parts in order to facilitate the binding and folding of the tag and the target protein of interest. Thanks to Phasin and Intein properties these parts can be used as high-specific TAGs for low-cost protein purification.<br />
<br />
At the moment were were not able to test Intein efficiency, but we could check if they affected the right folding of the target protein: we achieved this goal through the Silver Standard Assembly by using the GFP (<partinfo>BBa_K300005</partinfo>).<br />
<br />
These parts has been characterized respectively through:<br />
*pTet costitutive promoter devices:<br />
**<partinfo>BBa_K300088</partinfo><br />
**<partinfo>BBa_K300090</partinfo><br />
**<partinfo>BBa_K300099</partinfo><br />
*3OC6HSL inducible devices:<br />
**<partinfo>BBa_K300091</partinfo><br />
**<partinfo>BBa_K300092</partinfo><br />
and compared to a positive (<partinfo>BBa_K173000</partinfo>) and negative (<partinfo>BBa_B0031</partinfo>) control.<br />
<br />
==pTet costitutive promoter devices==<br />
===Methods===<br />
Inoculum (into 5 ml LB+Amp) from glycerol stock of:<br />
*<partinfo>BBa_K300088</partinfo><br />
*<partinfo>BBa_K300090</partinfo><br />
*<partinfo>BBa_K300099</partinfo><br />
*<partinfo>BBa_K173000</partinfo> (positive control)<br />
*<partinfo>BBa_B0031</partinfo> (negative control)<br />
They were let grow ON at +37°C, 220 rpm.<br />
<br />
The following day cultures were diluted 1:100 and let grow again for about five hours at +37°C, 220 rpm.<br />
<br />
Optical density (O.D.) of each cell culture was than measured with TECAN Infinte F200. Samples were diluted in order to obtain the same O.D. equal to 0,02.<br />
<br />
Than we performed a 21 hours' experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200. Each value shown is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted; cultures were shaken for 15 seconds every five minutes.<br />
<br />
===Results===<br />
<br />
{|align="center"<br />
|[[Image:UNIPV10_pTET_newP_ASB.png|thumb|300px|Raw growth curve]] || [[Image:UNIPV10_pTET_newP_GFP.png|thumb|300px|Raw GFP curve]]<br />
|}<br />
<table align="center"><br />
<tr><br />
<td><br />
[[Image:UNIPV10_pTET_newP_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<table border="1" align="center"><br />
<tr align="center"><br />
<th>Culture</th><th>Doubling time [min.] ± std error</th><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K173000</partinfo></td><td>76.3336 ± 1.4362</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300088</partinfo></td><td>74.8806 ± 2.7699</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300090</partinfo></td><td>75.9433 ± 3.6808</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300099</partinfo></td><td>78.4634 ± 2.5622</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_B0031</partinfo></td><td>70.8421 ± 2.2181</td><br />
</tr><br />
</table><br />
<br />
===Discussion===<br />
All cell cultures showed a similar growth curve and doubling time was computed as described [[Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation|here]] in order to have informations about the burden due to synthesis of such fusion proteins. It's possible to see that all doubling time are very similar; it's possible to assert that the expression of these BioBrick parts doesn't cause abnormal stress to the cells.<br />
<br />
In GFP curve it's possible to appreciate that in <partinfo>BBa_K300088</partinfo>, <partinfo>BBa_K300090</partinfo>, <partinfo>BBa_K300099</partinfo> GFP accumulation it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. These results show the right folding of the green fluorescent protein assembled downstream of the genetic circuit.<br />
<br />
The mean protein synthesis rate was also computed over the growth exponential phase, showing again an appreciable GFP production rate that is about a half of the positive control.<br />
<br />
==3OC6HSL inducible devices==<br />
===Methods===<br />
Inoculum (into 5 ml LB+Amp) from glycerol stock of:<br />
*<partinfo>BBa_K300091</partinfo><br />
*<partinfo>BBa_K300092</partinfo><br />
*<partinfo>BBa_K173000</partinfo> (positive control)<br />
*<partinfo>BBa_B0031</partinfo> (negative control)<br />
They were let grow ON at +37°C, 220 rpm.<br />
<br />
The following day cultures were diluted 1:100 and let grow again for about five hours at +37°C, 220 rpm.<br />
<br />
Optical density (O.D.) of each cell culture was than measured with TECAN Infinte F200. Samples were diluted in order to obtain the same O.D. equal to 0,02.<br />
<br />
Than we performed a 21 hours' experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200. <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> circuits were induced 100nM with HSL directly into multiplate well. Each value shown is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted; cultures were shaken for 15 seconds every five minutes.<br />
<br />
===Results===<br />
<table align="center"><br />
<tr><br />
<td>[[Image:UNIPV10_HSL_c_ASB.png|thumb|300px|Raw growth curve]]</td><br />
<td>[[Image:UNIPV10_HSL_c_GFP.png|thumb|300px|Raw GFP curve]]</td><br />
</tr><br />
</table><br />
<table align="center"><br />
<tr><br />
<td>[[Image:UNIPV10_HSL_c_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]</td><br />
</tr><br />
</table><br />
<br />
<table border="1" align="center"><br />
<tr align="center"><br />
<th>Culture</th><th>Doubling time [min.] ± std error</th><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K173000</partinfo></td><td>76.3336 ± 1.4362</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300091</partinfo><br/>induced</td><td>121.1434 ± 7.0275</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300091</partinfo><br/>not induced</td><td>74.4267 ± 1.3696</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300092</partinfo><br/>induced</td><td>122.6088 ± 1.2785</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300092</partinfo><br/>not induced</td><td>71.5105 ± 2.7113</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_B0031</partinfo></td><td>70.8421 ± 2.2181</td><br />
</tr><br />
</table><br />
<br />
===Discussion===<br />
All cell cultures showed a similar growth curve and doubling time was computed as described [[Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation|here]] in order to have informations about the burden due to synthesis of such fusion proteins. It's possible to see that all doubling time are very similar except for induced cultures. In this case doubling time is much higher than posite control and not induced cultures; so it's possible to assert that in this case there's a kind of metabolic burden higher than in the others, maybe because of the inducible system.<br />
<br />
In GFP curve it's possible to appreciate that in induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> GFP accumulation profile it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. On the other hand not induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> show a profile very similar to the last one. These results show the right folding of the green fluorescent protein assembled downstream of the genetic circuit and that the inducible system works as expected.<br />
<br />
The mean protein synthesis rate was also computed over the growth exponential phase, showing again an GFP production rate that is different from negative control. Not induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> show a low GFP synthesis rate maybe due to 3OC6HSL inducible circuit leakage activity.<br />
<br />
</td><br />
</tr></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAutoTeam:UNIPV-Pavia/Project/PromotoriAuto2010-10-27T17:50:05Z<p>Susanna: </p>
<hr />
<div>__NOTOC__<br />
{{UNIPV-Pavia/header}} <br />
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<p align="center" valign="top"><br><br />
<font size="5" face="Cataneo BT" align="center" valign="top">'''''ProteInProgress: a cellular assembly line for protein manufacturing'''''</font><br />
</p><br />
<hr><br />
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/motivation"><br />
<img src="https://static.igem.org/mediawiki/2010/b/be/UNIPV_Pavia_motivation2_BN.jpg" width="75px" height="75px" alt="Motivation" title="Motivation"/><br />
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<img src="https://static.igem.org/mediawiki/2010/7/77/UNIPV_Pavia_result.jpg" width="75px" height="75px" alt="Implementation & Results" title="Implementation & Results"/><br />
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/references"><br />
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<p align="center" valign="top"><br><br />
<font size="5" align="center" valign="top"><b>Implementation and Results: Self-Inducible promoters</b></font><br />
</p><br />
<br><br />
<hr><br />
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<table align="center" border="0" width="80%"><br />
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[[Team:UNIPV-Pavia/Project/PromotoriAuto|Self-inducible promoters]]<br />
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntColi"><br />
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<br><br />
[[Team:UNIPV-Pavia/Project/IntColi|Integrative standard vector for E. coli]]<br />
</td><br />
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntYeast"><br />
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<br><br />
[[Team:UNIPV-Pavia/Project/IntYeast|Integrative standard vector for yeast]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/FasinaInteina"><br />
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[[Team:UNIPV-Pavia/Project/FasinaInteina|Purification of proteins]]<br />
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</table><br />
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<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/><br />
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<br />
=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein production</b>===<br />
<br />
'''Experimental implementation:''' <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:<br />
<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>Strength (a.u.)<br>reported in the Registry</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 2547<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||1791<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||623<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||1185<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||844<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||256<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||396<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||1429<br />
|}<br />
<br />
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]], using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:<br />
*high copy number plasmids and LB;<br />
*high copy number plasmids and M9;<br />
*low copy number plasmids and M9.<br />
It was not possible to evaluate promoters activities in low copy number plasmids in LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2]]. Data were analyzed as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]]; <br />
<br />
'''Results''': results are shown here.<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI fragment of <partinfo>BBa_J61002</partinfo>-BBa_J231xx in <partinfo>pSB4C5</partinfo> vector, in order to transfer the promoter and the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
<br />
Doubling times were evaluated for the described cultures (HC stands for High Copy and LC stands for Low Copy):<br />
{| align='center' border='1'<br />
|rowspan='2'|<b>Promoter</b><br />
|colspan='3'| <b>doubling time [minutes]</b><br />
|-<br />
| LB in HC plasmid || M9 in HC plasmid || M9 in LC plasmid<br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 33.75 <br> ± <br> 1.34 || 82.53 <br> ± <br> 2.45 || 86.11 <br> ± <br> 4.45<br />
|-<br />
|<partinfo>BBa_J23101</partinfo> || 35.93 <br> ± <br> 0.62 || 82.68 <br> ± <br> 1.84 || 86.42 <br> ± <br> 1.91<br />
|-<br />
|<partinfo>BBa_J23105</partinfo> || 29.86 <br> ± <br> 0.33 || 63.09 <br> ± <br> 7.08 || 85.00 <br> ± <br> 5.13<br />
|-<br />
|<partinfo>BBa_J23106</partinfo> || 29.17 <br> ± <br> 0.96 || 68.11 <br> ± <br> 4.25 || 88.71 <br> ± <br> 0.90<br />
|-<br />
|<partinfo>BBa_J23110</partinfo> || 31.28 <br> ± <br> 0.42 || 67.52 <br> ± <br> 5.87 || 76.15 <br> ± <br> 2.16<br />
|-<br />
|<partinfo>BBa_J23114</partinfo> || 28.97 <br> ± <br> 0.49 || 59.44 <br> ± <br> 5.20 || 80.12 <br> ± <br> 0.95<br />
|-<br />
|<partinfo>BBa_J23116</partinfo> || 28.14 <br> ± <br> 0.25 || 72.74 <br> ± <br> 0.37 || 81.68 <br> ± <br> 3.08<br />
|-<br />
|<partinfo>BBa_J23118</partinfo> || 32.84 <br> ± <br> 0.31 || 73.64 <br> ± <br> 2.41 || 89.86 <br> ± <br> 2.93<br />
|}<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the tested conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced amounts of LuxI protein that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformants could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluorescent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced by autoinducer generators</b>===<br />
<br />
'''Experimental implementation:''' The autoinducer generators <partinfo>BBa_K300030</partinfo>, <partinfo>BBa_K300028</partinfo>, <partinfo>BBa_K300029</partinfo>, <partinfo>BBa_K300025</partinfo>, <partinfo>BBa_K300026</partinfo> and <partinfo>BBa_K300027</partinfo> were, thus, characterized by measuring the concentration of HSL released in the medium of cultures grown for 6 hours. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 biosensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results:''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table below:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced by autoinducer generators in high copy vector.]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> after a 6 hour cell growth is reported in Fig.9 and in the table below:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced by the autoinducer generators in low copy vector.]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) in high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized in tables.<br />
<br />
<br />
<div align='center>Sender/Receiver devices assembled as a unique BioBrick part on the same vector</div><br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender/Receiver Device Vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|}<br />
<br />
<div align='center>Sender devices assembled on low copy number vector and Receiver device on high copy number vector</div><br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center' width='80%'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|300px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|300px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|300px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|300px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]]<br />
*K_HSL is the HSL synthesis rate per cell, it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis to estimate the HSL synthesis rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double. It was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
<br>[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> *<br />
| 8.94 10^-17 <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> *<br />
| 7.53 10^-18 <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br>[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
<br />
'''Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br></td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300030.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
|Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br></td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300028.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300029</partinfo> (wiki name: I16) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300029.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.31 <br> *<br />
| 1.17 10^-16 <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300025.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300026.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300027.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 70%; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two self-inducible devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were realized and characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell and an algorithm was proposed, in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In figure, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported, both for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid). <br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the Signal Molucule production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by data. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate results stronger at the increase of promoter's strength. <br />
<br />
For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably due to the fact that too high synthesis rate of HSL are injurious for the cell. <br />
A range of RPU values for promoters that control the Sender device was identified. When the PoPs-in signal belongs to this range, the expected behaviour is confirmed by experimental data. <br />
The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices, able to start the production of a target protein at different O.D._start values, in any cellular growth phase.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/CharacterizationTeam:UNIPV-Pavia/Parts/Characterization2010-10-27T17:46:22Z<p>Susanna: /* Data Analysis to estimate the HSL synthesis rate per cell */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign=top><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" valign="top" width="100%"><tr><td align="justify" valign="top" style="padding:20px" width="70%" colspan="2"><br />
<html><p align="center"><font size="5"><b>CHARACTERIZATION</b></font></p></html><hr><br><br />
<br />
=Our Parts=<br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th></tr><br />
<!-- <tr><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
</tr> --><br />
</table><br><br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/NewParts |New Parts]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300000 - BioBrick integrative base vector for E. coli |BBa_K300000 - BioBrick integrative base vector for E. coli]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300001 - BioBrick integrative base vector for S. cerevisiae|BBa_K300001 - BioBrick integrative base vector for S. cerevisiae]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain|BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300010 - PoPS-based self-inducible device|BBa_K300010 - PoPS-based self-inducible device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification|BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification]]<br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts |Improved Parts]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300002 - Phasin (PhaP) - head domain|BBa_K300002 - Phasin (PhaP) - head domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300003 - Phasin (PhaP) - internal domain|BBa_K300003 - Phasin (PhaP) - internal domain]]<br />
<br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry |Existing Parts from the Registry]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - R6K Origin of replication|BBa_J61001 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - chloramphenicol resistance cassette|BBa_P1004 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - GFP fusion brick|BBa_K125500 - GFP fusion brick]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<hr><br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=Growth conditions=<br />
===Microplate reader experiments for self-inducible promoters - Protocol #1===<br />
<br />
* 8 ul of long term storage glycerol stock were inoculated in 1 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
* The grown cultures were then diluted 1:100 in 1 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.<br />
* Cultures were then pelletted (2000 rpm, 10 minutes) in order to eliminate the HSL produced during the growth.<br />
* Supernatants were discarded and the pellets were resuspended in 1 ml of LB or M9 + suitable antibiotic and transferred to a 1.5 ml tube<br />
* These cultures were diluted 1:1000 in 1 ml of LB or M9 + suitable antibiotic and aliquoted in a flat-bottom 96-well microplate in triplicate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects, see https://2009.igem.org/Team:UNIPV-Pavia/Methods_Materials/Evaporation). All the wells were filled with a 200 ul volume.<br />
* The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
** 37°C constant for all the experiment;<br />
** sampling time of 5 minutes;<br />
** fluorescence gain of 50;<br />
** O.D. filter was 600 nm;<br />
** GFP filters were 485nm (ex) / 540nm (em);<br />
** 15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
** Variable experiment duration time (from 3 to 24 hours).<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2===<br />
<br />
*8 ul of long term storage glycerol stock were inoculated in 5 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
*The grown cultures were then diluted 1:100 in 5 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.<br />
*These new cultures were diluted to an O.D.600 of 0.02 (measured with a TECAN F200 microplate reader on a 200 ul of volume per well; it is not equivalent to the 1 cm pathlength cuvette) in 2 ml LB or M9 + suitable antibiotic. In order to have the cultures at the desired O.D.600, the following dilution was performed:<br />
[[Image:UNIPV_Pavia_OD600_dil.png|500px|center]]<br />
*These new dilutions were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects in the frame). All the wells were filled with a 200 ul volume.<br />
*The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
**37°C constant for all the experiment;<br />
**sampling time of 5 minutes;<br />
**fluorescence gain of 50 or 70;<br />
**O.D. filter was 600 nm;<br />
**GFP filters were 485nm (ex) / 540nm (em);<br />
**RFP filters were 535nm (ex) / 620nm (em);<br />
**15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
**Experiment duration time: about 6 hours.<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for 3OC6-HSL quantification by means of <partinfo>BBa_T9002</partinfo> biosensor - Protocol #3===<br />
<br />
The aim of this experiment is the quantification of the 3OC6-HSL produced by cultures harbouring plasmids derived from <partinfo>BBa_K300009</partinfo>. These plasmids are BBa_J231xx-<partinfo>BBa_K300009</partinfo> assemblies, where BBa_J231xx are Anderson Promoter Collection library members. This assembled parts are 3OC6-HSL generators and one important parameter to be evaluated is the concentration of 3OC6-HSL produced.<br />
<br />
The "3OC6-HSL generator" culture was processed as follows:<br />
*<em>Preparation of 3OC6-HSL generating cultures:</em><br />
**8 ul of BBa_J231xx-<partinfo>BBa_K300009</partinfo> long term glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours. <br />
**The grown cultures were then diluted 1:100 in 5 ml of LB supplemented medium and incubated in the same conditions as before for 6 hours.<br />
**Each falcon was pelletted (2000 rpm, 10 minutes) and supernatants were collected and filtered (0.2 um), in order to eliminate bacterial residues. These supernatants contained the 3OC6-HSL at the concentration produced by cultures. These supernatants were used to "induce" <partinfo>BBa_T9002</partinfo> cultures, in order to quantify the [HSL]. They were conserved at -20°C until the next day.<br />
*<em>Growth and preparation of the biosensor culture of <partinfo>BBa_T9002</partinfo></em><br />
**8 ul of <partinfo>BBa_T9002</partinfo> long term glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
**The grown cultures were then diluted 1:100 in 5 ml of LB supplemented medium and incubated in the same conditions as before until they reached an O.D.600 of 0.07 (measured by Tecan INFINITE F200).<br />
**the required number (see below) of 200 ul aliquots of culture were transferred in each well of a 96-well microplate.<br />
*<em>Calibration curve</em><br />
**A calibration curve was obtained, by inducing in triplicate wells of <partinfo>BBa_T9002</partinfo> with different known HSL concentrations:<br />
***10 uM<br />
***1 uM<br />
***100 nM<br />
*** 50 nM<br />
*** 10 nM<br />
*** 5 nM<br />
***2 nM<br />
***1 nM<br />
*** 0,5 nM<br />
*** 0,1 nM<br />
*** 0 M<br />
**All inductions were performed adding 2ul of "inducer solution" (3OC6-HSL, Sigma Aldrich) at the proper concentration in 200 ul of culture.<br />
*<em>HSL quantification</em><br />
**2ul of supernatants prepared as described above were used to induce 200 ul of <partinfo>BBa_T9002</partinfo> cultures in triplicate. <br />
**If the amount of 3OC6-HSL present in the supernatant was not enough concentrated to trigger the induction of <partinfo>BBa_T9002</partinfo>, a bigger amount of supernatant (X ul) was used to induce the cultures (200-X ul of cultures). In this way, the calibration curve was obtained by using the same amount of <partinfo>BBa_T9002</partinfo> culture (200-X ul).<br />
**Fluorescence and absorbance were measured after 30 min from the induction with a Tecan Infinite F200 microplate reader and, using the information provided by the calibration curve, the amount of 3OC6-HSL present in every supernatant was evaluated (each reported fluorescence value was corrected with the O.D.600 of the culture).<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for <partinfo>BBa_F2620</partinfo> - Protocol #4===<br />
<br />
This protocol is identical to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|this one]], but in addition, after cultures were transferred in the 96-well microplate, 2ul of inducer solution at the proper concentration were added to each well, thus obtaining the final desired concentration.<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=Data Analysis=<br />
<br><br />
===Preliminary remarks===<br />
*All our growth curves have been obtained subtracting for each time sample the broth O.D.600 measurement from that of the culture; broth was considered in the same conditions of the culture (i.e. induced with the same inducer concentration and supplemented with the same antibiotic of the culture).<br />
<br />
*Fluorescence signals have been obtained subtracting for each time sample the fluorescent measurement of a non-fluorescent culture from that of the target culture. The non-fluorescent culture was considered in the same conditions of the culture of interest (e.g. induced with the same inducer concentration and with the same plasmid/antibiotic resistance, but without fluorescent reporter genes). This operation allows the removal, from the target fluorescent signal, of the "self-fluorescent" component and the fluorescence signal obtained is "blanked".<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Doubling time evaluation===<br />
The natural logarithm of the growth curves (processed according to the above section) was computed and the linear phase (corresponding to the bacterial exponential growth phase) was isolated by visual inspection. Then the linear regression was performed in order to estimate the slope of the line m. Finally the doubling time was estimated as d=ln(2)/m [minutes].<br />
<br />
In the case of multiple growth curves for a strain, the mean value of the processed curves was computed for each time sample before applying the above described procedure. <br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis for self-inducible promoters (initiation-treshold determination)===<br />
<br />
The task of this analysis is the evaluation of the initiation transcription point (in terms of absorbance) for self-inducible devices. The transition O.D.600 value is named, from now on, O.D.start.<br />
Data from three indipendent wells were averaged and blanked. O.D.600 signals were blanked as described in "Preliminary Remarks" section, while the fluorescence signal was blanked with the fluorescence of <partinfo>BBa_T9002</partinfo> part, assembled in the same plasmid of the considered promoter. This operation allows the removal from the fluorescence signal of the autofluorescence component and of the “leaky” component (due to the leakage of ''lux pR'' promoter in absence of the autoinducer molecule HSL).<br />
<br />
O.D.start was evaluated by computing the ''Scell'' (GFPmut3 synthesis rate per cell) signal for the desired self-inducible promoter.<br />
<br />
Scell was obtained by computing (1/O.D.600)*dGFP/dt, where O.D.600 and GFP are the normalized absorbance and fluorescence signals.<br />
<br />
The goal is the estimation of the critical O.D.600 value (O.D.start) at which the Scell significantly increases. Because Scell is a very noisy signal, a threshold value which takes into account the noise variability was proposed and it is described below.<br />
<br />
Two different signals, measured from independent samples of the same non-fluorescent culture in the same experiment, are considered: C1 and C2. The fluorescence signal of C1 and C2 (F_C1 and F_C2 respectively) can be thought as the addition of a “real signal” and a noise component.<br />
<br />
[[Image:UNIPV_Pavia_noise1.png|400px|center]]<br />
<br />
F_C1 and F_C2 have the same expected value and the same standard deviation, since they are two independent realizations of the same aleatory process: in fact, they are two time series acquired from the same cultures in the same growth conditions by the same instrument.<br />
Noise signal was computed as:<br />
<br />
[[Image:UNIPV_Pavia_noise2.png|150px|center]]<br />
<br />
An interesting signal is the Scell of ''N'' time series. It can be evaluated as the time derivative of ''N'', divided by O.D.600 of the culture. It has the behaviour shown in figure:<br />
<br />
[[Image:UNIPV_Pavia_rumoreNoiseScell.png|600px|center|Scell Noise]]<br />
It is fair to say that the noise of Scell is bigger for low O.D.600 values and its amplitude decreases dramatically for higher O.D.600 values (e.g.: O.D.600>0.1 measured with the TECAN Infinite F200 in a 96-well microplate).<br />
<br />
The O.D.600-dependent noise model proposed for the Scell signal is:<br />
<br />
[[Image:UNIPV_Pavia_ModelNoiseScell.png|300px|center|Scell noise model]]<br />
<br />
According to the formula reported above, it is possible to obtain the sigma_bar value as follows:<br />
#multiply the Scell time series values with the corresponding O.D.600;<br />
#the sigma_bar constant value can be estimated as the standard deviation of this signal.<br />
<br />
The Scell*O.D.600 signal behaviour is reported below:<br />
<br />
[[Image:UNIPV_Pavia_rumoreNoise.png|600px|center|Measurement Noise]]<br />
<br />
As this figure shows, the noise amplitude of this signal is no more O.D.600-dependent and the sigma_bar can be obtained.<br />
<br />
<br />
The evaluation of the O.D.start can be performed supposing that the transcription starts when the signal is significantly different from the noise. A threshold on the signal amplitude was estabilished as reported here:<br />
<br />
[[Image:UNIPV_Pavia_threshold.png|300px|center|variance threshold]]<br />
<br />
In order to make the method more robust, induction was considered only when 5 consecutive values exceeded the threshold amplitude.<br />
<br />
<br />
The operation of subtracting the two signals F_C1 and F_C2 is analogous to the “blanking” operation performed on data, as described previously. For, this reason, under the hypothesis that the signals have the same variance (this is a consistent hypothesis, since they are measured by the same instrument in the same experimental conditions), this argument can be extended to the “blanked” data considered in the processing. <br />
In particular, it is evident that the "blanked" signal is affected by the same noise described above and thus, the noise of Scell signal is analogous to the one described before.<br />
Thus, this threshold was used to compute the O.D.start for the cultures. This heuristic algorithm was implemented in Matlab and analysis results are reported in the “results” section.<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis to estimate the HSL synthesis rate per cell===<br />
<br />
<br />
The autoinducer synthesys rate per cell is a very important parameter to be evaluated in quorum sensing systems. <br />
In fact the knowledge of this parameter enables the rational design of cell-communication systems, such as the self-inducible devices studied in this project.<br />
<br />
A model based approach was proposed to estimate this interesting parameter. <br />
<br />
Under the hypotheses that:<br />
* no HSL is present at the beginning of the experiment - consistent hypothesis because a growth medium washing step is always performed before the experiment start in order to remove the HSL produced until that moment,<br />
* the HSL synthesis rate is not time-dependent (cells produce HSL at the same rate in each growth phase - this hypotesis simplifies the data analysis, but it should be further validated),<br />
* the half life of the autoinducer is much longer than the experiment duration (i.e. the degradation of the molecule is negligible - this hypothesis is consistent, since no HSL-degrading enzyme is present in ''E. coli''),<br />
<br />
an Ordinary Differential Equation (ODE) model which describes the HSL production in the growth media can be written:<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula1.png|250px]]<br />
|}<br />
where:<br />
* [HSL(t)] is the concentration of HSL molecule in the growth medium;<br />
* O.D.600(t) is the growth curve of the considered culture;<br />
* N is the Colony Forming Units (CFU) per O.D.600 unit (i.e. CFU in a well = O.D.600 * N)<br />
* K_HSL is the 3OC6-HSL synthesis rate per cell<br />
<br />
<br />
The analytical solution of this system is:<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula2.png|300px]]<br />
|}<br />
K_HSL can be estimated for t=t_bar, corresponding to the transcription initiation time, as reported below.<br />
<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula3.png|250px]]<br />
|}<br />
where:<br />
* N was estimated by counting TOP10 colonies obtained by plating serial dilutions of cultures with known O.D.600.<br />
* [HSL(t_bar)] was estimated as described in the section [[Data Analysis - minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>]]<br />
* the O.D.600 time series was measured in each experiment, so its time integral can be computed by trapezoidal numerical integration.<br />
<br />
N was estimated in LB and M9 media and values are reported below:<br />
<br />
{| border='1' align='center'<br />
| || &nbsp;&nbsp;&nbsp;'''LB'''&nbsp;&nbsp;&nbsp; || &nbsp;&nbsp;&nbsp;'''M9'''&nbsp;&nbsp;&nbsp;<br />
|-<br />
| &nbsp; &nbsp; &nbsp; '''N''' &nbsp; &nbsp; &nbsp;|| &nbsp;&nbsp;2,818 * 10^9 &nbsp;&nbsp;||&nbsp;&nbsp; 2,123 * 10^9 &nbsp;&nbsp;<br />
|}<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data Analysis - minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>===<br />
<br />
Cultures were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for BBa_F2620 - Protocol #4|this protocol]].<br />
<br />
Data from three indipendent wells were averaged and blanked as described in "Preliminary Remarks" section.<br />
<br />
Minimum induction required to activate ''lux pR'' was evaluated by a visual inspection of Scell signal<br />
<br />
<br />
[[Image:pv_MinimumInduction.png|330px|thumb|center|Minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>]]<br />
<br />
<br />
<div align='center'><br />
'''HSL(t_bar)''' : 0,04 nM<br />
</div><br />
HSL(t_bar) was evaluated only in M9 medium because it is a minimum autofluorescence growth medium and it provides the most accurate result in estimating a low fluorescence.<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis for RPU evaluation===<br />
<br />
The RPUs are standard units proposed by Kelly J. et al., 2008, in which the relative transcriptional strength of a promoter can be measured using a reference standard.<br />
<br />
RPUs have been computed as:<br />
[[Image:UNIPV_Pavia_RPU_formula.png|400px|center]]<br />
<br />
in which:<br />
* phi is the promoter of interest and J23101 is the reference standard promoter (taken from the Anderson Promoter Collection);<br />
* F is the blanked fluorescence of the culture, computed by subtracting for each time sample the fluorescence value of a negative control (a non-fluorescent culture). In our experiments, the TOP10 cells bearing BBa_B0032 or BBa_B0033 were usually used because they are RBSs and do not have expression systems for reporter genes;<br />
* ASB is the blanked absorbance (O.D.600) of the culture, computed as described in "Preliminary remarks" section. <br />
RPU measurement has the following advantages (under suitable conditions)<br />
* it is proportional to PoPS (Polymerase Per Second), a very important parameter that expresses the transcription rate of a promoter;<br />
* it uses a reference standard and so measurements can be compared between different laboratories. <br />
The hypotheses on which RPU theory is based can be found in Kelly J. et al., 2008, as well as all the mathematical steps. From our point of view, the main hypotheses that have to be satisfied are the following:<br />
* the reporter protein must have a half life higher than the experiment duration (we use GFPmut3 - <partinfo>BBa_E0040</partinfo> -, which has an estimated half life of at least 24 hours, or an engineered RFP - <partinfo>BBa_E1010</partinfo>, for which the half life has not been measured, but is qualitatively comparable with the GFP's);<br />
* strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in J23101 reference standard.<br />
* steady state must be valid, so (dF/dt)/ASB (proportional to the GFP synthesis rate per cell) must be constant.<br />
<br />
In order to compute the RPUs, the Scell signals ((dGFP/dt)/ASB)) of the promoter of interest and of the reference J23101 were averaged in the time interval corresponding to the exponential growth phase. The boundaries of exponential phase were identified with a visual inspection of the linear phase of the logarithmic growth curve.<br />
<br />
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<br />
</td></tr></table><br />
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[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
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[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
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[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]<br><br><br />
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[[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry |Existing Parts from the Registry]]<hr><br />
</td></tr></table> --></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAutoTeam:UNIPV-Pavia/Project/PromotoriAuto2010-10-27T17:42:35Z<p>Susanna: /* Quantification of the HSL produced by autoinducer generators */</p>
<hr />
<div>__NOTOC__<br />
{{UNIPV-Pavia/header}} <br />
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<font size="5" face="Cataneo BT" align="center" valign="top">'''''ProteInProgress: a cellular assembly line for protein manufacturing'''''</font><br />
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<font size="5" align="center" valign="top"><b>Implementation and Results: Self-Inducible promoters</b></font><br />
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[[Team:UNIPV-Pavia/Project/PromotoriAuto|Self-inducible promoters]]<br />
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[[Team:UNIPV-Pavia/Project/IntColi|Integrative standard vector for E. coli]]<br />
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[[Team:UNIPV-Pavia/Project/IntYeast|Integrative standard vector for yeast]]<br />
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/FasinaInteina"><br />
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[[Team:UNIPV-Pavia/Project/FasinaInteina|Purification of proteins]]<br />
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<br />
=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein production</b>===<br />
<br />
'''Experimental implementation:''' <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:<br />
<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>Strength (a.u.)<br>reported in the Registry</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 2547<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||1791<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||623<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||1185<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||844<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||256<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||396<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||1429<br />
|}<br />
<br />
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]], using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:<br />
*high copy number plasmids and LB;<br />
*high copy number plasmids and M9;<br />
*low copy number plasmids and M9.<br />
It was not possible to evaluate promoters activities in low copy number plasmids in LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2]]. Data were analyzed as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]]; <br />
<br />
'''Results''': results are shown here.<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI fragment of <partinfo>BBa_J61002</partinfo>-BBa_J231xx in <partinfo>pSB4C5</partinfo> vector, in order to transfer the promoter and the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
<br />
Doubling times were evaluated for the described cultures (HC stands for High Copy and LC stands for Low Copy):<br />
{| align='center' border='1'<br />
|rowspan='2'|<b>Promoter</b><br />
|colspan='3'| <b>doubling time [minutes]</b><br />
|-<br />
| LB in HC plasmid || M9 in HC plasmid || M9 in LC plasmid<br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 33.75 <br> ± <br> 1.34 || 82.53 <br> ± <br> 2.45 || 86.11 <br> ± <br> 4.45<br />
|-<br />
|<partinfo>BBa_J23101</partinfo> || 35.93 <br> ± <br> 0.62 || 82.68 <br> ± <br> 1.84 || 86.42 <br> ± <br> 1.91<br />
|-<br />
|<partinfo>BBa_J23105</partinfo> || 29.86 <br> ± <br> 0.33 || 63.09 <br> ± <br> 7.08 || 85.00 <br> ± <br> 5.13<br />
|-<br />
|<partinfo>BBa_J23106</partinfo> || 29.17 <br> ± <br> 0.96 || 68.11 <br> ± <br> 4.25 || 88.71 <br> ± <br> 0.90<br />
|-<br />
|<partinfo>BBa_J23110</partinfo> || 31.28 <br> ± <br> 0.42 || 67.52 <br> ± <br> 5.87 || 76.15 <br> ± <br> 2.16<br />
|-<br />
|<partinfo>BBa_J23114</partinfo> || 28.97 <br> ± <br> 0.49 || 59.44 <br> ± <br> 5.20 || 80.12 <br> ± <br> 0.95<br />
|-<br />
|<partinfo>BBa_J23116</partinfo> || 28.14 <br> ± <br> 0.25 || 72.74 <br> ± <br> 0.37 || 81.68 <br> ± <br> 3.08<br />
|-<br />
|<partinfo>BBa_J23118</partinfo> || 32.84 <br> ± <br> 0.31 || 73.64 <br> ± <br> 2.41 || 89.86 <br> ± <br> 2.93<br />
|}<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the tested conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced amounts of LuxI protein that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformants could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluorescent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced by autoinducer generators</b>===<br />
<br />
'''Experimental implementation:''' The autoinducer generators <partinfo>BBa_K300030</partinfo>, <partinfo>BBa_K300028</partinfo>, <partinfo>BBa_K300029</partinfo>, <partinfo>BBa_K300025</partinfo>, <partinfo>BBa_K300026</partinfo> and <partinfo>BBa_K300027</partinfo> were, thus, characterized by measuring the concentration of HSL released in the medium of cultures grown for 6 hours. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 biosensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results:''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table below:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced by autoinducer generators in high copy vector.]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> after a 6 hour cell growth is reported in Fig.9 and in the table below:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced by the autoinducer generators in low copy vector.]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) in high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized in tables.<br />
<br />
<br />
<div align='center>Sender/Receiver devices assembled as a unique BioBrick part on the same vector</div><br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender/Receiver Device Vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|}<br />
<br />
<div align='center>Sender devices assembled on low copy number vector and Receiver device on high copy number vector</div><br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center' width='80%'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|300px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|300px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|300px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|300px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]]<br />
*K_HSL is the HSL synthesis rate per cell, it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis to estimate the HSL synthesis rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double. It was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
<br>[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> *<br />
| 8.94 10^-17 <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> *<br />
| 7.53 10^-18 <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br>[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
<br />
'''Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br></td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300030.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
|Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br></td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300028.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300029</partinfo> (wiki name: I16) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300029.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.31 <br> *<br />
| 1.17 10^-16 <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300025.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300026.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300027.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 70%; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two self-inducible devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were realized and characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell and an algorithm was proposed, in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In figure, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported, both for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid). <br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the Signal Molucule production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by data. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate results stronger at the increase of promoter's strength. <br />
<br />
For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably due to the fact that too high synthesis rate of HSL are injurious for the cell. <br />
A range of RPU values for promoters that control the Sender device was identified. When the PoPs-in signal belongs to this range, the expected behaviour is confirmed by experimental data. <br />
The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices, able to start the production of a target protein at different O.D._start values, in any cellular growth phase.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAutoTeam:UNIPV-Pavia/Project/PromotoriAuto2010-10-27T17:36:20Z<p>Susanna: </p>
<hr />
<div>__NOTOC__<br />
{{UNIPV-Pavia/header}} <br />
<br />
<br />
<table width="100%" border="0" align="center"><br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td align="justify" valign="top" style="padding:20px"><br />
<p align="center" valign="top"><br><br />
<font size="5" face="Cataneo BT" align="center" valign="top">'''''ProteInProgress: a cellular assembly line for protein manufacturing'''''</font><br />
</p><br />
<hr><br />
<br><br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/motivation"><br />
<img src="https://static.igem.org/mediawiki/2010/b/be/UNIPV_Pavia_motivation2_BN.jpg" width="75px" height="75px" alt="Motivation" title="Motivation"/><br />
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<img src="https://static.igem.org/mediawiki/2010/c/c8/UNIPV_Pavia_solution2_BN.jpg" width="75px" height="75px" alt="Solutions" title="Solutions"/></a><br />
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<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/results"><br />
<img src="https://static.igem.org/mediawiki/2010/7/77/UNIPV_Pavia_result.jpg" width="75px" height="75px" alt="Implementation & Results" title="Implementation & Results"/><br />
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<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/references"><br />
<img src="https://static.igem.org/mediawiki/2010/6/61/UNIPV_Paviareferences_BN.jpg" width="75px" height="75px" alt="References" title="References"/></a><br />
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</table><br />
<br />
<html><a name="indice"/></a></html><br />
<br />
<p align="center" valign="top"><br><br />
<font size="5" align="center" valign="top"><b>Implementation and Results: Self-Inducible promoters</b></font><br />
</p><br />
<br><br />
<hr><br />
<br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAuto"><br />
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<br><br />
[[Team:UNIPV-Pavia/Project/PromotoriAuto|Self-inducible promoters]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntColi"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/UNIPV_Pavia_Icona_intColi.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntColi|Integrative standard vector for E. coli]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntYeast"><br />
<img src="https://static.igem.org/mediawiki/2010/0/03/UNIPV_Pavia_Icona_IntYeast.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntYeast|Integrative standard vector for yeast]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/FasinaInteina"><br />
<img src="https://static.igem.org/mediawiki/2010/7/74/UNIPV_Pavia_Icona_Bioplastica.GIF" width="75px" height="75px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/FasinaInteina|Purification of proteins]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/><br />
</html><br />
<br />
=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein production</b>===<br />
<br />
'''Experimental implementation:''' <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:<br />
<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>Strength (a.u.)<br>reported in the Registry</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 2547<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||1791<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||623<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||1185<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||844<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||256<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||396<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||1429<br />
|}<br />
<br />
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]], using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:<br />
*high copy number plasmids and LB;<br />
*high copy number plasmids and M9;<br />
*low copy number plasmids and M9.<br />
It was not possible to evaluate promoters activities in low copy number plasmids in LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2]]. Data were analyzed as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]]; <br />
<br />
'''Results''': results are shown here.<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI fragment of <partinfo>BBa_J61002</partinfo>-BBa_J231xx in <partinfo>pSB4C5</partinfo> vector, in order to transfer the promoter and the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
<br />
Doubling times were evaluated for the described cultures (HC stands for High Copy and LC stands for Low Copy):<br />
{| align='center' border='1'<br />
|rowspan='2'|<b>Promoter</b><br />
|colspan='3'| <b>doubling time [minutes]</b><br />
|-<br />
| LB in HC plasmid || M9 in HC plasmid || M9 in LC plasmid<br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 33.75 <br> ± <br> 1.34 || 82.53 <br> ± <br> 2.45 || 86.11 <br> ± <br> 4.45<br />
|-<br />
|<partinfo>BBa_J23101</partinfo> || 35.93 <br> ± <br> 0.62 || 82.68 <br> ± <br> 1.84 || 86.42 <br> ± <br> 1.91<br />
|-<br />
|<partinfo>BBa_J23105</partinfo> || 29.86 <br> ± <br> 0.33 || 63.09 <br> ± <br> 7.08 || 85.00 <br> ± <br> 5.13<br />
|-<br />
|<partinfo>BBa_J23106</partinfo> || 29.17 <br> ± <br> 0.96 || 68.11 <br> ± <br> 4.25 || 88.71 <br> ± <br> 0.90<br />
|-<br />
|<partinfo>BBa_J23110</partinfo> || 31.28 <br> ± <br> 0.42 || 67.52 <br> ± <br> 5.87 || 76.15 <br> ± <br> 2.16<br />
|-<br />
|<partinfo>BBa_J23114</partinfo> || 28.97 <br> ± <br> 0.49 || 59.44 <br> ± <br> 5.20 || 80.12 <br> ± <br> 0.95<br />
|-<br />
|<partinfo>BBa_J23116</partinfo> || 28.14 <br> ± <br> 0.25 || 72.74 <br> ± <br> 0.37 || 81.68 <br> ± <br> 3.08<br />
|-<br />
|<partinfo>BBa_J23118</partinfo> || 32.84 <br> ± <br> 0.31 || 73.64 <br> ± <br> 2.41 || 89.86 <br> ± <br> 2.93<br />
|}<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the tested conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced amounts of LuxI protein that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformants could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluorescent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced by autoinducer generators</b>===<br />
<br />
'''Experimental implementation:''' The autoinducer generators <partinfo>BBa_K300030</partinfo>, <partinfo>BBa_K300028</partinfo>, <partinfo>BBa_K300029</partinfo>, <partinfo>BBa_K300025</partinfo>, <partinfo>BBa_K300026</partinfo> and <partinfo>BBa_K300027</partinfo> were, thus, characterized by measuring the concentration of HSL released in the medium of cultures grown for 6 hours. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 sensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results:''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table below:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced by autoinducer generators in high copy vector.]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> after a 6 hour cell growth is reported in Fig.9 and in the table below:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced by the autoinducer generators in low copy vector.]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) in high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized in tables.<br />
<br />
<br />
<div align='center>Sender/Receiver devices assembled as a unique BioBrick part on the same vector</div><br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender/Receiver Device Vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|}<br />
<br />
<div align='center>Sender devices assembled on low copy number vector and Receiver device on high copy number vector</div><br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center' width='80%'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|300px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|300px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|300px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|300px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]]<br />
*K_HSL is the HSL synthesis rate per cell, it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis to estimate the HSL synthesis rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double. It was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
<br>[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> *<br />
| 8.94 10^-17 <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> *<br />
| 7.53 10^-18 <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br>[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
<br />
'''Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br></td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300030.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
|Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br></td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300028.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300029</partinfo> (wiki name: I16) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300029.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.31 <br> *<br />
| 1.17 10^-16 <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300025.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300026.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300027.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 70%; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two self-inducible devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were realized and characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell and an algorithm was proposed, in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In figure, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported, both for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid). <br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the Signal Molucule production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by data. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate results stronger at the increase of promoter's strength. <br />
<br />
For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably due to the fact that too high synthesis rate of HSL are injurious for the cell. <br />
A range of RPU values for promoters that control the Sender device was identified. When the PoPs-in signal belongs to this range, the expected behaviour is confirmed by experimental data. <br />
The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices, able to start the production of a target protein at different O.D._start values, in any cellular growth phase.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/CharacterizationTeam:UNIPV-Pavia/Parts/Characterization2010-10-27T17:22:51Z<p>Susanna: /* Existing Parts from the Registry */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign=top><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" valign="top" width="100%"><tr><td align="justify" valign="top" style="padding:20px" width="70%" colspan="2"><br />
<html><p align="center"><font size="5"><b>CHARACTERIZATION</b></font></p></html><hr><br><br />
<br />
=Our Parts=<br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th></tr><br />
<!-- <tr><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
</tr> --><br />
</table><br><br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/NewParts |New Parts]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300000 - BioBrick integrative base vector for E. coli |BBa_K300000 - BioBrick integrative base vector for E. coli]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300001 - BioBrick integrative base vector for S. cerevisiae|BBa_K300001 - BioBrick integrative base vector for S. cerevisiae]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain|BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300010 - PoPS-based self-inducible device|BBa_K300010 - PoPS-based self-inducible device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification|BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification]]<br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts |Improved Parts]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300002 - Phasin (PhaP) - head domain|BBa_K300002 - Phasin (PhaP) - head domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300003 - Phasin (PhaP) - internal domain|BBa_K300003 - Phasin (PhaP) - internal domain]]<br />
<br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry |Existing Parts from the Registry]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - R6K Origin of replication|BBa_J61001 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - chloramphenicol resistance cassette|BBa_P1004 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - GFP fusion brick|BBa_K125500 - GFP fusion brick]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<hr><br><br />
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=Growth conditions=<br />
===Microplate reader experiments for self-inducible promoters - Protocol #1===<br />
<br />
* 8 ul of long term storage glycerol stock were inoculated in 1 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
* The grown cultures were then diluted 1:100 in 1 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.<br />
* Cultures were then pelletted (2000 rpm, 10 minutes) in order to eliminate the HSL produced during the growth.<br />
* Supernatants were discarded and the pellets were resuspended in 1 ml of LB or M9 + suitable antibiotic and transferred to a 1.5 ml tube<br />
* These cultures were diluted 1:1000 in 1 ml of LB or M9 + suitable antibiotic and aliquoted in a flat-bottom 96-well microplate in triplicate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects, see https://2009.igem.org/Team:UNIPV-Pavia/Methods_Materials/Evaporation). All the wells were filled with a 200 ul volume.<br />
* The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
** 37°C constant for all the experiment;<br />
** sampling time of 5 minutes;<br />
** fluorescence gain of 50;<br />
** O.D. filter was 600 nm;<br />
** GFP filters were 485nm (ex) / 540nm (em);<br />
** 15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
** Variable experiment duration time (from 3 to 24 hours).<br />
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===Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2===<br />
<br />
*8 ul of long term storage glycerol stock were inoculated in 5 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
*The grown cultures were then diluted 1:100 in 5 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.<br />
*These new cultures were diluted to an O.D.600 of 0.02 (measured with a TECAN F200 microplate reader on a 200 ul of volume per well; it is not equivalent to the 1 cm pathlength cuvette) in 2 ml LB or M9 + suitable antibiotic. In order to have the cultures at the desired O.D.600, the following dilution was performed:<br />
[[Image:UNIPV_Pavia_OD600_dil.png|500px|center]]<br />
*These new dilutions were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects in the frame). All the wells were filled with a 200 ul volume.<br />
*The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
**37°C constant for all the experiment;<br />
**sampling time of 5 minutes;<br />
**fluorescence gain of 50 or 70;<br />
**O.D. filter was 600 nm;<br />
**GFP filters were 485nm (ex) / 540nm (em);<br />
**RFP filters were 535nm (ex) / 620nm (em);<br />
**15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
**Experiment duration time: about 6 hours.<br />
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===Microplate reader experiments for 3OC6-HSL quantification by means of <partinfo>BBa_T9002</partinfo> biosensor - Protocol #3===<br />
<br />
The aim of this experiment is the quantification of the 3OC6-HSL produced by cultures harbouring plasmids derived from <partinfo>BBa_K300009</partinfo>. These plasmids are BBa_J231xx-<partinfo>BBa_K300009</partinfo> assemblies, where BBa_J231xx are Anderson Promoter Collection library members. This assembled parts are 3OC6-HSL generators and one important parameter to be evaluated is the concentration of 3OC6-HSL produced.<br />
<br />
The "3OC6-HSL generator" culture was processed as follows:<br />
*<em>Preparation of 3OC6-HSL generating cultures:</em><br />
**8 ul of BBa_J231xx-<partinfo>BBa_K300009</partinfo> long term glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours. <br />
**The grown cultures were then diluted 1:100 in 5 ml of LB supplemented medium and incubated in the same conditions as before for 6 hours.<br />
**Each falcon was pelletted (2000 rpm, 10 minutes) and supernatants were collected and filtered (0.2 um), in order to eliminate bacterial residues. These supernatants contained the 3OC6-HSL at the concentration produced by cultures. These supernatants were used to "induce" <partinfo>BBa_T9002</partinfo> cultures, in order to quantify the [HSL]. They were conserved at -20°C until the next day.<br />
*<em>Growth and preparation of the biosensor culture of <partinfo>BBa_T9002</partinfo></em><br />
**8 ul of <partinfo>BBa_T9002</partinfo> long term glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
**The grown cultures were then diluted 1:100 in 5 ml of LB supplemented medium and incubated in the same conditions as before until they reached an O.D.600 of 0.07 (measured by Tecan INFINITE F200).<br />
**the required number (see below) of 200 ul aliquots of culture were transferred in each well of a 96-well microplate.<br />
*<em>Calibration curve</em><br />
**A calibration curve was obtained, by inducing in triplicate wells of <partinfo>BBa_T9002</partinfo> with different known HSL concentrations:<br />
***10 uM<br />
***1 uM<br />
***100 nM<br />
*** 50 nM<br />
*** 10 nM<br />
*** 5 nM<br />
***2 nM<br />
***1 nM<br />
*** 0,5 nM<br />
*** 0,1 nM<br />
*** 0 M<br />
**All inductions were performed adding 2ul of "inducer solution" (3OC6-HSL, Sigma Aldrich) at the proper concentration in 200 ul of culture.<br />
*<em>HSL quantification</em><br />
**2ul of supernatants prepared as described above were used to induce 200 ul of <partinfo>BBa_T9002</partinfo> cultures in triplicate. <br />
**If the amount of 3OC6-HSL present in the supernatant was not enough concentrated to trigger the induction of <partinfo>BBa_T9002</partinfo>, a bigger amount of supernatant (X ul) was used to induce the cultures (200-X ul of cultures). In this way, the calibration curve was obtained by using the same amount of <partinfo>BBa_T9002</partinfo> culture (200-X ul).<br />
**Fluorescence and absorbance were measured after 30 min from the induction with a Tecan Infinite F200 microplate reader and, using the information provided by the calibration curve, the amount of 3OC6-HSL present in every supernatant was evaluated (each reported fluorescence value was corrected with the O.D.600 of the culture).<br />
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===Microplate reader experiments for <partinfo>BBa_F2620</partinfo> - Protocol #4===<br />
<br />
This protocol is identical to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|this one]], but in addition, after cultures were transferred in the 96-well microplate, 2ul of inducer solution at the proper concentration were added to each well, thus obtaining the final desired concentration.<br />
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=Data Analysis=<br />
<br><br />
===Preliminary remarks===<br />
*All our growth curves have been obtained subtracting for each time sample the broth O.D.600 measurement from that of the culture; broth was considered in the same conditions of the culture (e.g. induced with the same inducer concentration and supplemented with the same antibiotic of the culture).<br />
<br />
*Fluorescence signals have been obtained subtracting for each time sample the fluorescent measurement of a non-fluorescent culture from that of the target culture. The non-fluorescent culture was considered in the same conditions of the target (e.g. induced with the same inducer concentration and with the BioBrick carried by the same plasmid, not encoding a fluorescent protein). This operation allows the removal, from the target fluorescent signal, of the "self-fluorescent" component and the fluorescence signal obtained is "blanked".<br />
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===Doubling time evaluation===<br />
The natural logarithm of the growth curves (processed according to the above section) was computed and the linear phase (corresponding to the bacterial exponential growth phase) was isolated by visual inspection. Then the linear regression was performed in order to estimate the slope of the line m. Finally the doubling time was estimated as d=ln(2)/m [minutes].<br />
<br />
In the case of multiple growth curves for a strain, the mean value of the processed curves was computed for each time sample before applying the above described procedure. <br />
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===Data analysis for self-inducible promoters (initiation-treshold determination)===<br />
<br />
The task of this analysis is the evaluation of the initiation transcription point (in terms of absorbance) for self-inducible devices. The transition O.D.600 value is named, from now on, O.D.start.<br />
Data from three indipendent wells were averaged and blanked. O.D.600 signals were blanked as described in "Preliminary Remarks" section, while the fluorescence signal was blanked with the fluorescence of <partinfo>BBa_T9002</partinfo> part, assembled in the same plasmid of the considered promoter. This operation allows the removal from the fluorescence signal of the autofluorescence component and of the “leaky” component (due to the leakage of ''lux pR'' promoter in absence of the autoinducer molecule HSL).<br />
<br />
O.D.start was evaluated by computing the ''Scell'' (GFPmut3 synthesis rate per cell) signal for the desired self-inducible promoter.<br />
<br />
Scell was obtained by computing (1/O.D.600)*dGFP/dt, where O.D.600 and GFP are the normalized absorbance and fluorescence signals.<br />
<br />
The goal is the estimation of the critical O.D.600 value (O.D.start) at which the Scell significantly increases. Because Scell is a very noisy signal, a threshold value which takes into account the noise variability was proposed and it is described below.<br />
<br />
Two different signals, measured from independent samples of the same non-fluorescent culture in the same experiment, are considered: C1 and C2. The fluorescence signal of C1 and C2 (F_C1 and F_C2 respectively) can be thought as the addition of a “real signal” and a noise component.<br />
<br />
[[Image:UNIPV_Pavia_noise1.png|400px|center]]<br />
<br />
F_C1 and F_C2 have the same expected value and the same standard deviation, since they are two independent realizations of the same aleatory process: in fact, they are two time series acquired from the same cultures in the same growth conditions by the same instrument.<br />
Noise signal was computed as:<br />
<br />
[[Image:UNIPV_Pavia_noise2.png|150px|center]]<br />
<br />
An interesting signal is the Scell of ''N'' time series. It can be evaluated as the time derivative of ''N'', divided by O.D.600 of the culture. It has the behaviour shown in figure:<br />
<br />
[[Image:UNIPV_Pavia_rumoreNoiseScell.png|600px|center|Scell Noise]]<br />
It is fair to say that the noise of Scell is bigger for low O.D.600 values and its amplitude decreases dramatically for higher O.D.600 values (e.g.: O.D.600>0.1 measured with the TECAN Infinite F200 in a 96-well microplate).<br />
<br />
The O.D.600-dependent noise model proposed for the Scell signal is:<br />
<br />
[[Image:UNIPV_Pavia_ModelNoiseScell.png|300px|center|Scell noise model]]<br />
<br />
According to the formula reported above, it is possible to obtain the sigma_bar value as follows:<br />
#multiply the Scell time series values with the corresponding O.D.600;<br />
#the sigma_bar constant value can be estimated as the standard deviation of this signal.<br />
<br />
The Scell*O.D.600 signal behaviour is reported below:<br />
<br />
[[Image:UNIPV_Pavia_rumoreNoise.png|600px|center|Measurement Noise]]<br />
<br />
As this figure shows, the noise amplitude of this signal is no more O.D.600-dependent and the sigma_bar can be obtained.<br />
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The evaluation of the O.D.start can be performed supposing that the transcription starts when the signal is significantly different from the noise. A threshold on the signal amplitude was estabilished as reported here:<br />
<br />
[[Image:UNIPV_Pavia_threshold.png|300px|center|variance threshold]]<br />
<br />
In order to make the method more robust, induction was considered only when 5 consecutive values exceeded the threshold amplitude.<br />
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The operation of subtracting the two signals F_C1 and F_C2 is analogous to the “blanking” operation performed on data, as described previously. For, this reason, under the hypothesis that the signals have the same variance (this is a consistent hypothesis, since they are measured by the same instrument in the same experimental conditions), this argument can be extended to the “blanked” data considered in the processing. <br />
In particular, it is evident that the "blanked" signal is affected by the same noise described above and thus, the noise of Scell signal is analogous to the one described before.<br />
Thus, this threshold was used to compute the O.D.start for the cultures. This heuristic algorithm was implemented in Matlab and analysis results are reported in the “results” section.<br />
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===Data Analysis to estimate the HSL synthesis rate per cell===<br />
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The autoinducer synthesys rate per cell is a very important parameter to be evaluated in quorum sensing systems. <br />
In fact the knowledge of this parameter enables the rational design of cell-communication systems, such as the self inducible devices studied in this project.<br />
<br />
A model based approach was proposed to estimate this interesting parameter. <br />
<br />
Under the hypotheses that:<br />
* no HSL is present at the beginning of the experiment - consistent hypothesis because a growth medium washing step is always performed before the experiment start in order to remove the HSL produced until that moment,<br />
* the HSL synthesis rate is not time-dependent (cells produce HSL at the same rate in each growth phase - this hypotesis simplifies the data analysis, but it should be further validated),<br />
* the half life of the autoinducer is much longer than the experiment duration (i.e. the degradation of the molecule is negligible - this hypothesis is consistent, since no HSL-degrading enzyme is present in ''E. coli''),<br />
<br />
an Ordinary Differential Equation (ODE) model which describes the HSL production in the growth media can be written:<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula1.png|250px]]<br />
|}<br />
where:<br />
* [HSL(t)] is the concentration of HSL molecule in the growth medium;<br />
* O.D.600(t) is the growth curve of the considered culture;<br />
* N is the Colony Forming Units (CFU) per O.D.600 unit (i.e. CFU in a well = O.D.600 * N)<br />
* K_HSL is the 3OC6-HSL synthesis rate per cell<br />
<br />
<br />
The analytical solution of this system is:<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula2.png|300px]]<br />
|}<br />
K_HSL can be estimated for t=t_bar, corresponding to the trasncription initiation time, as reported below.<br />
<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula3.png|250px]]<br />
|}<br />
where:<br />
* N was estimated by counting TOP10 colonies obtained by plating serial dilutions of cultures with known O.D.600.<br />
* [HSL(t_bar)] was estimated as described in the section [[Data Analysis - minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>]]<br />
* the O.D.600 time series was measured in each experiment, so its time integral can be computed by trapezoidal numerical integration.<br />
<br />
N was estimated and values are reported below:<br />
<br />
{| border='1' align='center'<br />
| || &nbsp;&nbsp;&nbsp;'''LB'''&nbsp;&nbsp;&nbsp; || &nbsp;&nbsp;&nbsp;'''M9'''&nbsp;&nbsp;&nbsp;<br />
|-<br />
| &nbsp; &nbsp; &nbsp; '''N''' &nbsp; &nbsp; &nbsp;|| &nbsp;&nbsp;2,818 * 10^9 &nbsp;&nbsp;||&nbsp;&nbsp; 2,123 * 10^9 &nbsp;&nbsp;<br />
|}<br />
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===Data Analysis - minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>===<br />
<br />
Cultures were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for BBa_F2620 - Protocol #4|this protocol]].<br />
<br />
Data from three indipendent wells were averaged and blanked as described in "Preliminary Remarks" section.<br />
<br />
Minimum induction required to activate ''lux pR'' was evaluated by a visual inspection of Scell signal<br />
<br />
<br />
[[Image:pv_MinimumInduction.png|330px|thumb|center|Minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>]]<br />
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<br />
<div align='center'><br />
'''HSL(t_bar)''' : 0,04 nM<br />
</div><br />
HSL(t_bar) was evaluated only in M9 medium because it is a minimum autofluorescence growth medium and it provides the most accurate result in estimating a low fluorescence.<br />
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===Data analysis for RPU evaluation===<br />
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The RPUs are standard units proposed by Kelly J. et al., 2008, in which the relative transcriptional strength of a promoter can be measured using a reference standard.<br />
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RPUs have been computed as:<br />
[[Image:UNIPV_Pavia_RPU_formula.png|400px|center]]<br />
<br />
in which:<br />
* phi is the promoter of interest and J23101 is the reference standard promoter (taken from the Anderson Promoter Collection);<br />
* F is the blanked fluorescence of the culture, computed by subtracting for each time sample the fluorescence value of a negative control (a non-fluorescent culture). In our experiments, the TOP10 cells bearing BBa_B0032 or BBa_B0033 were usually used because they are RBSs and do not have expression systems for reporter genes;<br />
* ASB is the blanked absorbance (O.D.600) of the culture, computed as described in "Preliminary remarks" section. <br />
RPU measurement has the following advantages (under suitable conditions)<br />
* it is proportional to PoPS (Polymerase Per Second), a very important parameter that expresses the transcription rate of a promoter;<br />
* it uses a reference standard and so measurements can be compared between different laboratories. <br />
The hypotheses on which RPU theory is based can be found in Kelly J. et al., 2008, as well as all the mathematical steps. From our point of view, the main hypotheses that have to be satisfied are the following:<br />
* the reporter protein must have a half life higher than the experiment duration (we use GFPmut3 - <partinfo>BBa_E0040</partinfo> -, which has an estimated half life of at least 24 hours, or an engineered RFP - <partinfo>BBa_E1010</partinfo>, for which the half life has not been measured, but is qualitatively comparable with the GFP's);<br />
* strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in J23101 reference standard.<br />
* steady state must be valid, so (dF/dt)/ASB (proportional to the GFP synthesis rate per cell) must be constant.<br />
<br />
In order to compute the RPUs, the Scell signals ((dGFP/dt)/ASB)) of the promoter of interest and of the reference J23101 were averaged in the time interval corresponding to the exponential growth phase. The boundaries of exponential phase were identified with a visual inspection of the linear phase of the logarithmic growth curve.<br />
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[[Team:UNIPV-Pavia/Parts/Characterization/NewParts |New Parts]]<hr><br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
</td></tr><br />
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[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]<br><br><br />
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<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts |Improved Parts]]<hr><br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry |Existing Parts from the Registry]]<hr><br />
</td></tr></table> --></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistryTeam:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry2010-10-27T17:20:20Z<p>Susanna: /* BBa_K125500 - chloramphenicol resistance cassette */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><td valign=top width="70%"><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<html><p align="center"><font size="4"><b>EXISTING PARTS FROM THE REGISTRY</b></font></p></html><hr><br />
<br />
<tr><td width="100%"><br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><br />
<td align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization]<br />
</td><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th><br />
</tr><br />
</table><br><br />
<br />
=Existing Parts from the Registry: list=<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - R6K Origin of replication|BBa_J61001 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J2311 - constitutive promoters from Anderson's collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - chloramphenicol resistance cassette|BBa_P1004 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - GFP fusion brick|BBa_K125500 - GFP fusion brick]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=<partinfo>BBa_K300009</partinfo>/<partinfo>BBa_I4102</partinfo> - PoPS->3OC6HSL sender device=<br />
=<partinfo>BBa_F2620</partinfo> - 3OC6HSL -> PoPS Receiver=<br />
=<partinfo>BBa_J61001</partinfo> - R6K Origin of replication=<br />
<br />
=<partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> - constitutive promoters from Anderson's collection=<br />
<br />
=<partinfo>BBa_P1004</partinfo> - chloramphenicol resistance cassette=<br />
<br />
=<partinfo>BBa_K125500</partinfo> - GFP fusion brick=<br />
<br />
=<partinfo>BBa_J72008</partinfo> - phi80 integration helper plasmid pInt80-649=<br />
<br />
<tr><td><br></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistryTeam:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry2010-10-27T17:19:40Z<p>Susanna: /* BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoter collection */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><td valign=top width="70%"><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<html><p align="center"><font size="4"><b>EXISTING PARTS FROM THE REGISTRY</b></font></p></html><hr><br />
<br />
<tr><td width="100%"><br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><br />
<td align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization]<br />
</td><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th><br />
</tr><br />
</table><br><br />
<br />
=Existing Parts from the Registry: list=<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - R6K Origin of replication|BBa_J61001 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J2311 - constitutive promoters from Anderson's collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - chloramphenicol resistance cassette|BBa_P1004 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - GFP fusion brick|BBa_K125500 - GFP fusion brick]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=<partinfo>BBa_K300009</partinfo>/<partinfo>BBa_I4102</partinfo> - PoPS->3OC6HSL sender device=<br />
=<partinfo>BBa_F2620</partinfo> - 3OC6HSL -> PoPS Receiver=<br />
=<partinfo>BBa_J61001</partinfo> - R6K Origin of replication=<br />
<br />
=<partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> - constitutive promoters from Anderson's collection=<br />
<br />
=<partinfo>BBa_P1004</partinfo> - chloramphenicol resistance cassette=<br />
<br />
=<partinfo>BBa_K125500</partinfo> - chloramphenicol resistance cassette=<br />
=<partinfo>BBa_J72008</partinfo> - phi80 integration helper plasmid pInt80-649=<br />
<br />
<tr><td><br></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistryTeam:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry2010-10-27T17:18:54Z<p>Susanna: /* Existing Parts from the Registry: list */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><td valign=top width="70%"><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<html><p align="center"><font size="4"><b>EXISTING PARTS FROM THE REGISTRY</b></font></p></html><hr><br />
<br />
<tr><td width="100%"><br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><br />
<td align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization]<br />
</td><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th><br />
</tr><br />
</table><br><br />
<br />
=Existing Parts from the Registry: list=<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - R6K Origin of replication|BBa_J61001 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J2311 - constitutive promoters from Anderson's collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - chloramphenicol resistance cassette|BBa_P1004 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - GFP fusion brick|BBa_K125500 - GFP fusion brick]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=<partinfo>BBa_K300009</partinfo>/<partinfo>BBa_I4102</partinfo> - PoPS->3OC6HSL sender device=<br />
=<partinfo>BBa_F2620</partinfo> - 3OC6HSL -> PoPS Receiver=<br />
=<partinfo>BBa_J61001</partinfo> - R6K Origin of replication=<br />
<br />
=<partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> - constitutive Anderson promoter collection=<br />
=<partinfo>BBa_P1004</partinfo> - chloramphenicol resistance cassette=<br />
<br />
=<partinfo>BBa_K125500</partinfo> - chloramphenicol resistance cassette=<br />
=<partinfo>BBa_J72008</partinfo> - phi80 integration helper plasmid pInt80-649=<br />
<br />
<tr><td><br></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/CharacterizationTeam:UNIPV-Pavia/Parts/Characterization2010-10-27T17:16:51Z<p>Susanna: /* Existing Parts from the Registry */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign=top><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" valign="top" width="100%"><tr><td align="justify" valign="top" style="padding:20px" width="70%" colspan="2"><br />
<html><p align="center"><font size="5"><b>CHARACTERIZATION</b></font></p></html><hr><br><br />
<br />
=Our Parts=<br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th></tr><br />
<!-- <tr><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
</tr> --><br />
</table><br><br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/NewParts |New Parts]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300000 - BioBrick integrative base vector for E. coli |BBa_K300000 - BioBrick integrative base vector for E. coli]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300001 - BioBrick integrative base vector for S. cerevisiae|BBa_K300001 - BioBrick integrative base vector for S. cerevisiae]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain|BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300010 - PoPS-based self-inducible device|BBa_K300010 - PoPS-based self-inducible device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification|BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification]]<br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts |Improved Parts]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300002 - Phasin (PhaP) - head domain|BBa_K300002 - Phasin (PhaP) - head domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300003 - Phasin (PhaP) - internal domain|BBa_K300003 - Phasin (PhaP) - internal domain]]<br />
<br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry |Existing Parts from the Registry]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - R6K Origin of replication|BBa_J61001 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive promoters from Anderson's collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - chloramphenicol resistance cassette|BBa_P1004 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 -GFP fusion brick|BBa_K125500 - GFP fusion brick]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<hr><br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=Growth conditions=<br />
===Microplate reader experiments for self-inducible promoters - Protocol #1===<br />
<br />
* 8 ul of long term storage glycerol stock were inoculated in 1 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
* The grown cultures were then diluted 1:100 in 1 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.<br />
* Cultures were then pelletted (2000 rpm, 10 minutes) in order to eliminate the HSL produced during the growth.<br />
* Supernatants were discarded and the pellets were resuspended in 1 ml of LB or M9 + suitable antibiotic and transferred to a 1.5 ml tube<br />
* These cultures were diluted 1:1000 in 1 ml of LB or M9 + suitable antibiotic and aliquoted in a flat-bottom 96-well microplate in triplicate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects, see https://2009.igem.org/Team:UNIPV-Pavia/Methods_Materials/Evaporation). All the wells were filled with a 200 ul volume.<br />
* The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
** 37°C constant for all the experiment;<br />
** sampling time of 5 minutes;<br />
** fluorescence gain of 50;<br />
** O.D. filter was 600 nm;<br />
** GFP filters were 485nm (ex) / 540nm (em);<br />
** 15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
** Variable experiment duration time (from 3 to 24 hours).<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2===<br />
<br />
*8 ul of long term storage glycerol stock were inoculated in 5 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
*The grown cultures were then diluted 1:100 in 5 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.<br />
*These new cultures were diluted to an O.D.600 of 0.02 (measured with a TECAN F200 microplate reader on a 200 ul of volume per well; it is not equivalent to the 1 cm pathlength cuvette) in 2 ml LB or M9 + suitable antibiotic. In order to have the cultures at the desired O.D.600, the following dilution was performed:<br />
[[Image:UNIPV_Pavia_OD600_dil.png|500px|center]]<br />
*These new dilutions were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects in the frame). All the wells were filled with a 200 ul volume.<br />
*The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
**37°C constant for all the experiment;<br />
**sampling time of 5 minutes;<br />
**fluorescence gain of 50 or 70;<br />
**O.D. filter was 600 nm;<br />
**GFP filters were 485nm (ex) / 540nm (em);<br />
**RFP filters were 535nm (ex) / 620nm (em);<br />
**15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
**Experiment duration time: about 6 hours.<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for 3OC6-HSL quantification by means of <partinfo>BBa_T9002</partinfo> biosensor - Protocol #3===<br />
<br />
The aim of this experiment is the quantification of the 3OC6-HSL produced by cultures harbouring plasmids derived from <partinfo>BBa_K300009</partinfo>. These plasmids are BBa_J231xx-<partinfo>BBa_K300009</partinfo> assemblies, where BBa_J231xx are Anderson Promoter Collection library members. This assembled parts are 3OC6-HSL generators and one important parameter to be evaluated is the concentration of 3OC6-HSL produced.<br />
<br />
The "3OC6-HSL generator" culture was processed as follows:<br />
*<em>Preparation of 3OC6-HSL generating cultures:</em><br />
**8 ul of BBa_J231xx-<partinfo>BBa_K300009</partinfo> long term glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours. <br />
**The grown cultures were then diluted 1:100 in 5 ml of LB supplemented medium and incubated in the same conditions as before for 6 hours.<br />
**Each falcon was pelletted (2000 rpm, 10 minutes) and supernatants were collected and filtered (0.2 um), in order to eliminate bacterial residues. These supernatants contained the 3OC6-HSL at the concentration produced by cultures. These supernatants were used to "induce" <partinfo>BBa_T9002</partinfo> cultures, in order to quantify the [HSL]. They were conserved at -20°C until the next day.<br />
*<em>Growth and preparation of the biosensor culture of <partinfo>BBa_T9002</partinfo></em><br />
**8 ul of <partinfo>BBa_T9002</partinfo> long term glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
**The grown cultures were then diluted 1:100 in 5 ml of LB supplemented medium and incubated in the same conditions as before until they reached an O.D.600 of 0.07 (measured by Tecan INFINITE F200).<br />
**the required number (see below) of 200 ul aliquots of culture were transferred in each well of a 96-well microplate.<br />
*<em>Calibration curve</em><br />
**A calibration curve was obtained, by inducing in triplicate wells of <partinfo>BBa_T9002</partinfo> with different known HSL concentrations:<br />
***10 uM<br />
***1 uM<br />
***100 nM<br />
*** 50 nM<br />
*** 10 nM<br />
*** 5 nM<br />
***2 nM<br />
***1 nM<br />
*** 0,5 nM<br />
*** 0,1 nM<br />
*** 0 M<br />
**All inductions were performed adding 2ul of "inducer solution" (3OC6-HSL, Sigma Aldrich) at the proper concentration in 200 ul of culture.<br />
*<em>HSL quantification</em><br />
**2ul of supernatants prepared as described above were used to induce 200 ul of <partinfo>BBa_T9002</partinfo> cultures in triplicate. <br />
**If the amount of 3OC6-HSL present in the supernatant was not enough concentrated to trigger the induction of <partinfo>BBa_T9002</partinfo>, a bigger amount of supernatant (X ul) was used to induce the cultures (200-X ul of cultures). In this way, the calibration curve was obtained by using the same amount of <partinfo>BBa_T9002</partinfo> culture (200-X ul).<br />
**Fluorescence and absorbance were measured after 30 min from the induction with a Tecan Infinite F200 microplate reader and, using the information provided by the calibration curve, the amount of 3OC6-HSL present in every supernatant was evaluated (each reported fluorescence value was corrected with the O.D.600 of the culture).<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for <partinfo>BBa_F2620</partinfo> - Protocol #4===<br />
<br />
This protocol is identical to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|this one]], but in addition, after cultures were transferred in the 96-well microplate, 2ul of inducer solution at the proper concentration were added to each well, thus obtaining the final desired concentration.<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=Data Analysis=<br />
<br><br />
===Preliminary remarks===<br />
*All our growth curves have been obtained subtracting for each time sample the broth O.D.600 measurement from that of the culture; broth was considered in the same conditions of the culture (e.g. induced with the same inducer concentration and supplemented with the same antibiotic of the culture).<br />
<br />
*Fluorescence signals have been obtained subtracting for each time sample the fluorescent measurement of a non-fluorescent culture from that of the target culture. The non-fluorescent culture was considered in the same conditions of the target (e.g. induced with the same inducer concentration and with the BioBrick carried by the same plasmid, not encoding a fluorescent protein). This operation allows the removal, from the target fluorescent signal, of the "self-fluorescent" component and the fluorescence signal obtained is "blanked".<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Doubling time evaluation===<br />
The natural logarithm of the growth curves (processed according to the above section) was computed and the linear phase (corresponding to the bacterial exponential growth phase) was isolated by visual inspection. Then the linear regression was performed in order to estimate the slope of the line m. Finally the doubling time was estimated as d=ln(2)/m [minutes].<br />
<br />
In the case of multiple growth curves for a strain, the mean value of the processed curves was computed for each time sample before applying the above described procedure. <br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis for self-inducible promoters (initiation-treshold determination)===<br />
<br />
The task of this analysis is the evaluation of the initiation transcription point (in terms of absorbance) for self-inducible devices. The transition O.D.600 value is named, from now on, O.D.start.<br />
Data from three indipendent wells were averaged and blanked. O.D.600 signals were blanked as described in "Preliminary Remarks" section, while the fluorescence signal was blanked with the fluorescence of <partinfo>BBa_T9002</partinfo> part, assembled in the same plasmid of the considered promoter. This operation allows the removal from the fluorescence signal of the autofluorescence component and of the “leaky” component (due to the leakage of ''lux pR'' promoter in absence of the autoinducer molecule HSL).<br />
<br />
O.D.start was evaluated by computing the ''Scell'' (GFPmut3 synthesis rate per cell) signal for the desired self-inducible promoter.<br />
<br />
Scell was obtained by computing (1/O.D.600)*dGFP/dt, where O.D.600 and GFP are the normalized absorbance and fluorescence signals.<br />
<br />
The goal is the estimation of the critical O.D.600 value (O.D.start) at which the Scell significantly increases. Because Scell is a very noisy signal, a threshold value which takes into account the noise variability was proposed and it is described below.<br />
<br />
Two different signals, measured from independent samples of the same non-fluorescent culture in the same experiment, are considered: C1 and C2. The fluorescence signal of C1 and C2 (F_C1 and F_C2 respectively) can be thought as the addition of a “real signal” and a noise component.<br />
<br />
[[Image:UNIPV_Pavia_noise1.png|400px|center]]<br />
<br />
F_C1 and F_C2 have the same expected value and the same standard deviation, since they are two independent realizations of the same aleatory process: in fact, they are two time series acquired from the same cultures in the same growth conditions by the same instrument.<br />
Noise signal was computed as:<br />
<br />
[[Image:UNIPV_Pavia_noise2.png|150px|center]]<br />
<br />
An interesting signal is the Scell of ''N'' time series. It can be evaluated as the time derivative of ''N'', divided by O.D.600 of the culture. It has the behaviour shown in figure:<br />
<br />
[[Image:UNIPV_Pavia_rumoreNoiseScell.png|600px|center|Scell Noise]]<br />
It is fair to say that the noise of Scell is bigger for low O.D.600 values and its amplitude decreases dramatically for higher O.D.600 values (e.g.: O.D.600>0.1 measured with the TECAN Infinite F200 in a 96-well microplate).<br />
<br />
The O.D.600-dependent noise model proposed for the Scell signal is:<br />
<br />
[[Image:UNIPV_Pavia_ModelNoiseScell.png|300px|center|Scell noise model]]<br />
<br />
According to the formula reported above, it is possible to obtain the sigma_bar value as follows:<br />
#multiply the Scell time series values with the corresponding O.D.600;<br />
#the sigma_bar constant value can be estimated as the standard deviation of this signal.<br />
<br />
The Scell*O.D.600 signal behaviour is reported below:<br />
<br />
[[Image:UNIPV_Pavia_rumoreNoise.png|600px|center|Measurement Noise]]<br />
<br />
As this figure shows, the noise amplitude of this signal is no more O.D.600-dependent and the sigma_bar can be obtained.<br />
<br />
<br />
The evaluation of the O.D.start can be performed supposing that the transcription starts when the signal is significantly different from the noise. A threshold on the signal amplitude was estabilished as reported here:<br />
<br />
[[Image:UNIPV_Pavia_threshold.png|300px|center|variance threshold]]<br />
<br />
In order to make the method more robust, induction was considered only when 5 consecutive values exceeded the threshold amplitude.<br />
<br />
<br />
The operation of subtracting the two signals F_C1 and F_C2 is analogous to the “blanking” operation performed on data, as described previously. For, this reason, under the hypothesis that the signals have the same variance (this is a consistent hypothesis, since they are measured by the same instrument in the same experimental conditions), this argument can be extended to the “blanked” data considered in the processing. <br />
In particular, it is evident that the "blanked" signal is affected by the same noise described above and thus, the noise of Scell signal is analogous to the one described before.<br />
Thus, this threshold was used to compute the O.D.start for the cultures. This heuristic algorithm was implemented in Matlab and analysis results are reported in the “results” section.<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data Analysis to estimate the HSL synthesis rate per cell===<br />
<br />
<br />
The autoinducer synthesys rate per cell is a very important parameter to be evaluated in quorum sensing systems. <br />
In fact the knowledge of this parameter enables the rational design of cell-communication systems, such as the self inducible devices studied in this project.<br />
<br />
A model based approach was proposed to estimate this interesting parameter. <br />
<br />
Under the hypotheses that:<br />
* no HSL is present at the beginning of the experiment - consistent hypothesis because a growth medium washing step is always performed before the experiment start in order to remove the HSL produced until that moment,<br />
* the HSL synthesis rate is not time-dependent (cells produce HSL at the same rate in each growth phase - this hypotesis simplifies the data analysis, but it should be further validated),<br />
* the half life of the autoinducer is much longer than the experiment duration (i.e. the degradation of the molecule is negligible - this hypothesis is consistent, since no HSL-degrading enzyme is present in ''E. coli''),<br />
<br />
an Ordinary Differential Equation (ODE) model which describes the HSL production in the growth media can be written:<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula1.png|250px]]<br />
|}<br />
where:<br />
* [HSL(t)] is the concentration of HSL molecule in the growth medium;<br />
* O.D.600(t) is the growth curve of the considered culture;<br />
* N is the Colony Forming Units (CFU) per O.D.600 unit (i.e. CFU in a well = O.D.600 * N)<br />
* K_HSL is the 3OC6-HSL synthesis rate per cell<br />
<br />
<br />
The analytical solution of this system is:<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula2.png|300px]]<br />
|}<br />
K_HSL can be estimated for t=t_bar, corresponding to the trasncription initiation time, as reported below.<br />
<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula3.png|250px]]<br />
|}<br />
where:<br />
* N was estimated by counting TOP10 colonies obtained by plating serial dilutions of cultures with known O.D.600.<br />
* [HSL(t_bar)] was estimated as described in the section [[Data Analysis - minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>]]<br />
* the O.D.600 time series was measured in each experiment, so its time integral can be computed by trapezoidal numerical integration.<br />
<br />
N was estimated and values are reported below:<br />
<br />
{| border='1' align='center'<br />
| || &nbsp;&nbsp;&nbsp;'''LB'''&nbsp;&nbsp;&nbsp; || &nbsp;&nbsp;&nbsp;'''M9'''&nbsp;&nbsp;&nbsp;<br />
|-<br />
| &nbsp; &nbsp; &nbsp; '''N''' &nbsp; &nbsp; &nbsp;|| &nbsp;&nbsp;2,818 * 10^9 &nbsp;&nbsp;||&nbsp;&nbsp; 2,123 * 10^9 &nbsp;&nbsp;<br />
|}<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data Analysis - minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>===<br />
<br />
Cultures were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for BBa_F2620 - Protocol #4|this protocol]].<br />
<br />
Data from three indipendent wells were averaged and blanked as described in "Preliminary Remarks" section.<br />
<br />
Minimum induction required to activate ''lux pR'' was evaluated by a visual inspection of Scell signal<br />
<br />
<br />
[[Image:pv_MinimumInduction.png|330px|thumb|center|Minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>]]<br />
<br />
<br />
<div align='center'><br />
'''HSL(t_bar)''' : 0,04 nM<br />
</div><br />
HSL(t_bar) was evaluated only in M9 medium because it is a minimum autofluorescence growth medium and it provides the most accurate result in estimating a low fluorescence.<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis for RPU evaluation===<br />
<br />
The RPUs are standard units proposed by Kelly J. et al., 2008, in which the relative transcriptional strength of a promoter can be measured using a reference standard.<br />
<br />
RPUs have been computed as:<br />
[[Image:UNIPV_Pavia_RPU_formula.png|400px|center]]<br />
<br />
in which:<br />
* phi is the promoter of interest and J23101 is the reference standard promoter (taken from the Anderson Promoter Collection);<br />
* F is the blanked fluorescence of the culture, computed by subtracting for each time sample the fluorescence value of a negative control (a non-fluorescent culture). In our experiments, the TOP10 cells bearing BBa_B0032 or BBa_B0033 were usually used because they are RBSs and do not have expression systems for reporter genes;<br />
* ASB is the blanked absorbance (O.D.600) of the culture, computed as described in "Preliminary remarks" section. <br />
RPU measurement has the following advantages (under suitable conditions)<br />
* it is proportional to PoPS (Polymerase Per Second), a very important parameter that expresses the transcription rate of a promoter;<br />
* it uses a reference standard and so measurements can be compared between different laboratories. <br />
The hypotheses on which RPU theory is based can be found in Kelly J. et al., 2008, as well as all the mathematical steps. From our point of view, the main hypotheses that have to be satisfied are the following:<br />
* the reporter protein must have a half life higher than the experiment duration (we use GFPmut3 - <partinfo>BBa_E0040</partinfo> -, which has an estimated half life of at least 24 hours, or an engineered RFP - <partinfo>BBa_E1010</partinfo>, for which the half life has not been measured, but is qualitatively comparable with the GFP's);<br />
* strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in J23101 reference standard.<br />
* steady state must be valid, so (dF/dt)/ASB (proportional to the GFP synthesis rate per cell) must be constant.<br />
<br />
In order to compute the RPUs, the Scell signals ((dGFP/dt)/ASB)) of the promoter of interest and of the reference J23101 were averaged in the time interval corresponding to the exponential growth phase. The boundaries of exponential phase were identified with a visual inspection of the linear phase of the logarithmic growth curve.<br />
<br />
<br><br />
----<br />
<br><br />
<br />
</td></tr></table><br />
<br />
<!-- <td valign="top"><br />
<font class="menu"><br />
<table border="0" width="80px" align="center" class="menu"><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts |New Parts]]<hr><br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]<br><br><br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts |Improved Parts]]<hr><br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry |Existing Parts from the Registry]]<hr><br />
</td></tr></table> --></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/CharacterizationTeam:UNIPV-Pavia/Parts/Characterization2010-10-27T17:15:42Z<p>Susanna: /* Existing Parts from the Registry */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign=top><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" valign="top" width="100%"><tr><td align="justify" valign="top" style="padding:20px" width="70%" colspan="2"><br />
<html><p align="center"><font size="5"><b>CHARACTERIZATION</b></font></p></html><hr><br><br />
<br />
=Our Parts=<br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th></tr><br />
<!-- <tr><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
</tr> --><br />
</table><br><br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/NewParts |New Parts]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300000 - BioBrick integrative base vector for E. coli |BBa_K300000 - BioBrick integrative base vector for E. coli]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300001 - BioBrick integrative base vector for S. cerevisiae|BBa_K300001 - BioBrick integrative base vector for S. cerevisiae]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain|BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300010 - PoPS-based self-inducible device|BBa_K300010 - PoPS-based self-inducible device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification|BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification]]<br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts |Improved Parts]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300002 - Phasin (PhaP) - head domain|BBa_K300002 - Phasin (PhaP) - head domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300003 - Phasin (PhaP) - internal domain|BBa_K300003 - Phasin (PhaP) - internal domain]]<br />
<br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry |Existing Parts from the Registry]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - R6K Origin of replication|BBa_J61001 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoter collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoters collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - chloramphenicol resistance cassette|BBa_P1004 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 -GFP fusion brick|BBa_K125500 - GFP fusion brick]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<hr><br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=Growth conditions=<br />
===Microplate reader experiments for self-inducible promoters - Protocol #1===<br />
<br />
* 8 ul of long term storage glycerol stock were inoculated in 1 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
* The grown cultures were then diluted 1:100 in 1 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.<br />
* Cultures were then pelletted (2000 rpm, 10 minutes) in order to eliminate the HSL produced during the growth.<br />
* Supernatants were discarded and the pellets were resuspended in 1 ml of LB or M9 + suitable antibiotic and transferred to a 1.5 ml tube<br />
* These cultures were diluted 1:1000 in 1 ml of LB or M9 + suitable antibiotic and aliquoted in a flat-bottom 96-well microplate in triplicate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects, see https://2009.igem.org/Team:UNIPV-Pavia/Methods_Materials/Evaporation). All the wells were filled with a 200 ul volume.<br />
* The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
** 37°C constant for all the experiment;<br />
** sampling time of 5 minutes;<br />
** fluorescence gain of 50;<br />
** O.D. filter was 600 nm;<br />
** GFP filters were 485nm (ex) / 540nm (em);<br />
** 15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
** Variable experiment duration time (from 3 to 24 hours).<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2===<br />
<br />
*8 ul of long term storage glycerol stock were inoculated in 5 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
*The grown cultures were then diluted 1:100 in 5 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.<br />
*These new cultures were diluted to an O.D.600 of 0.02 (measured with a TECAN F200 microplate reader on a 200 ul of volume per well; it is not equivalent to the 1 cm pathlength cuvette) in 2 ml LB or M9 + suitable antibiotic. In order to have the cultures at the desired O.D.600, the following dilution was performed:<br />
[[Image:UNIPV_Pavia_OD600_dil.png|500px|center]]<br />
*These new dilutions were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects in the frame). All the wells were filled with a 200 ul volume.<br />
*The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
**37°C constant for all the experiment;<br />
**sampling time of 5 minutes;<br />
**fluorescence gain of 50 or 70;<br />
**O.D. filter was 600 nm;<br />
**GFP filters were 485nm (ex) / 540nm (em);<br />
**RFP filters were 535nm (ex) / 620nm (em);<br />
**15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
**Experiment duration time: about 6 hours.<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for 3OC6-HSL quantification by means of <partinfo>BBa_T9002</partinfo> biosensor - Protocol #3===<br />
<br />
The aim of this experiment is the quantification of the 3OC6-HSL produced by cultures harbouring plasmids derived from <partinfo>BBa_K300009</partinfo>. These plasmids are BBa_J231xx-<partinfo>BBa_K300009</partinfo> assemblies, where BBa_J231xx are Anderson Promoter Collection library members. This assembled parts are 3OC6-HSL generators and one important parameter to be evaluated is the concentration of 3OC6-HSL produced.<br />
<br />
The "3OC6-HSL generator" culture was processed as follows:<br />
*<em>Preparation of 3OC6-HSL generating cultures:</em><br />
**8 ul of BBa_J231xx-<partinfo>BBa_K300009</partinfo> long term glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours. <br />
**The grown cultures were then diluted 1:100 in 5 ml of LB supplemented medium and incubated in the same conditions as before for 6 hours.<br />
**Each falcon was pelletted (2000 rpm, 10 minutes) and supernatants were collected and filtered (0.2 um), in order to eliminate bacterial residues. These supernatants contained the 3OC6-HSL at the concentration produced by cultures. These supernatants were used to "induce" <partinfo>BBa_T9002</partinfo> cultures, in order to quantify the [HSL]. They were conserved at -20°C until the next day.<br />
*<em>Growth and preparation of the biosensor culture of <partinfo>BBa_T9002</partinfo></em><br />
**8 ul of <partinfo>BBa_T9002</partinfo> long term glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
**The grown cultures were then diluted 1:100 in 5 ml of LB supplemented medium and incubated in the same conditions as before until they reached an O.D.600 of 0.07 (measured by Tecan INFINITE F200).<br />
**the required number (see below) of 200 ul aliquots of culture were transferred in each well of a 96-well microplate.<br />
*<em>Calibration curve</em><br />
**A calibration curve was obtained, by inducing in triplicate wells of <partinfo>BBa_T9002</partinfo> with different known HSL concentrations:<br />
***10 uM<br />
***1 uM<br />
***100 nM<br />
*** 50 nM<br />
*** 10 nM<br />
*** 5 nM<br />
***2 nM<br />
***1 nM<br />
*** 0,5 nM<br />
*** 0,1 nM<br />
*** 0 M<br />
**All inductions were performed adding 2ul of "inducer solution" (3OC6-HSL, Sigma Aldrich) at the proper concentration in 200 ul of culture.<br />
*<em>HSL quantification</em><br />
**2ul of supernatants prepared as described above were used to induce 200 ul of <partinfo>BBa_T9002</partinfo> cultures in triplicate. <br />
**If the amount of 3OC6-HSL present in the supernatant was not enough concentrated to trigger the induction of <partinfo>BBa_T9002</partinfo>, a bigger amount of supernatant (X ul) was used to induce the cultures (200-X ul of cultures). In this way, the calibration curve was obtained by using the same amount of <partinfo>BBa_T9002</partinfo> culture (200-X ul).<br />
**Fluorescence and absorbance were measured after 30 min from the induction with a Tecan Infinite F200 microplate reader and, using the information provided by the calibration curve, the amount of 3OC6-HSL present in every supernatant was evaluated (each reported fluorescence value was corrected with the O.D.600 of the culture).<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for <partinfo>BBa_F2620</partinfo> - Protocol #4===<br />
<br />
This protocol is identical to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|this one]], but in addition, after cultures were transferred in the 96-well microplate, 2ul of inducer solution at the proper concentration were added to each well, thus obtaining the final desired concentration.<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=Data Analysis=<br />
<br><br />
===Preliminary remarks===<br />
*All our growth curves have been obtained subtracting for each time sample the broth O.D.600 measurement from that of the culture; broth was considered in the same conditions of the culture (e.g. induced with the same inducer concentration and supplemented with the same antibiotic of the culture).<br />
<br />
*Fluorescence signals have been obtained subtracting for each time sample the fluorescent measurement of a non-fluorescent culture from that of the target culture. The non-fluorescent culture was considered in the same conditions of the target (e.g. induced with the same inducer concentration and with the BioBrick carried by the same plasmid, not encoding a fluorescent protein). This operation allows the removal, from the target fluorescent signal, of the "self-fluorescent" component and the fluorescence signal obtained is "blanked".<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Doubling time evaluation===<br />
The natural logarithm of the growth curves (processed according to the above section) was computed and the linear phase (corresponding to the bacterial exponential growth phase) was isolated by visual inspection. Then the linear regression was performed in order to estimate the slope of the line m. Finally the doubling time was estimated as d=ln(2)/m [minutes].<br />
<br />
In the case of multiple growth curves for a strain, the mean value of the processed curves was computed for each time sample before applying the above described procedure. <br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis for self-inducible promoters (initiation-treshold determination)===<br />
<br />
The task of this analysis is the evaluation of the initiation transcription point (in terms of absorbance) for self-inducible devices. The transition O.D.600 value is named, from now on, O.D.start.<br />
Data from three indipendent wells were averaged and blanked. O.D.600 signals were blanked as described in "Preliminary Remarks" section, while the fluorescence signal was blanked with the fluorescence of <partinfo>BBa_T9002</partinfo> part, assembled in the same plasmid of the considered promoter. This operation allows the removal from the fluorescence signal of the autofluorescence component and of the “leaky” component (due to the leakage of ''lux pR'' promoter in absence of the autoinducer molecule HSL).<br />
<br />
O.D.start was evaluated by computing the ''Scell'' (GFPmut3 synthesis rate per cell) signal for the desired self-inducible promoter.<br />
<br />
Scell was obtained by computing (1/O.D.600)*dGFP/dt, where O.D.600 and GFP are the normalized absorbance and fluorescence signals.<br />
<br />
The goal is the estimation of the critical O.D.600 value (O.D.start) at which the Scell significantly increases. Because Scell is a very noisy signal, a threshold value which takes into account the noise variability was proposed and it is described below.<br />
<br />
Two different signals, measured from independent samples of the same non-fluorescent culture in the same experiment, are considered: C1 and C2. The fluorescence signal of C1 and C2 (F_C1 and F_C2 respectively) can be thought as the addition of a “real signal” and a noise component.<br />
<br />
[[Image:UNIPV_Pavia_noise1.png|400px|center]]<br />
<br />
F_C1 and F_C2 have the same expected value and the same standard deviation, since they are two independent realizations of the same aleatory process: in fact, they are two time series acquired from the same cultures in the same growth conditions by the same instrument.<br />
Noise signal was computed as:<br />
<br />
[[Image:UNIPV_Pavia_noise2.png|150px|center]]<br />
<br />
An interesting signal is the Scell of ''N'' time series. It can be evaluated as the time derivative of ''N'', divided by O.D.600 of the culture. It has the behaviour shown in figure:<br />
<br />
[[Image:UNIPV_Pavia_rumoreNoiseScell.png|600px|center|Scell Noise]]<br />
It is fair to say that the noise of Scell is bigger for low O.D.600 values and its amplitude decreases dramatically for higher O.D.600 values (e.g.: O.D.600>0.1 measured with the TECAN Infinite F200 in a 96-well microplate).<br />
<br />
The O.D.600-dependent noise model proposed for the Scell signal is:<br />
<br />
[[Image:UNIPV_Pavia_ModelNoiseScell.png|300px|center|Scell noise model]]<br />
<br />
According to the formula reported above, it is possible to obtain the sigma_bar value as follows:<br />
#multiply the Scell time series values with the corresponding O.D.600;<br />
#the sigma_bar constant value can be estimated as the standard deviation of this signal.<br />
<br />
The Scell*O.D.600 signal behaviour is reported below:<br />
<br />
[[Image:UNIPV_Pavia_rumoreNoise.png|600px|center|Measurement Noise]]<br />
<br />
As this figure shows, the noise amplitude of this signal is no more O.D.600-dependent and the sigma_bar can be obtained.<br />
<br />
<br />
The evaluation of the O.D.start can be performed supposing that the transcription starts when the signal is significantly different from the noise. A threshold on the signal amplitude was estabilished as reported here:<br />
<br />
[[Image:UNIPV_Pavia_threshold.png|300px|center|variance threshold]]<br />
<br />
In order to make the method more robust, induction was considered only when 5 consecutive values exceeded the threshold amplitude.<br />
<br />
<br />
The operation of subtracting the two signals F_C1 and F_C2 is analogous to the “blanking” operation performed on data, as described previously. For, this reason, under the hypothesis that the signals have the same variance (this is a consistent hypothesis, since they are measured by the same instrument in the same experimental conditions), this argument can be extended to the “blanked” data considered in the processing. <br />
In particular, it is evident that the "blanked" signal is affected by the same noise described above and thus, the noise of Scell signal is analogous to the one described before.<br />
Thus, this threshold was used to compute the O.D.start for the cultures. This heuristic algorithm was implemented in Matlab and analysis results are reported in the “results” section.<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data Analysis to estimate the HSL synthesis rate per cell===<br />
<br />
<br />
The autoinducer synthesys rate per cell is a very important parameter to be evaluated in quorum sensing systems. <br />
In fact the knowledge of this parameter enables the rational design of cell-communication systems, such as the self inducible devices studied in this project.<br />
<br />
A model based approach was proposed to estimate this interesting parameter. <br />
<br />
Under the hypotheses that:<br />
* no HSL is present at the beginning of the experiment - consistent hypothesis because a growth medium washing step is always performed before the experiment start in order to remove the HSL produced until that moment,<br />
* the HSL synthesis rate is not time-dependent (cells produce HSL at the same rate in each growth phase - this hypotesis simplifies the data analysis, but it should be further validated),<br />
* the half life of the autoinducer is much longer than the experiment duration (i.e. the degradation of the molecule is negligible - this hypothesis is consistent, since no HSL-degrading enzyme is present in ''E. coli''),<br />
<br />
an Ordinary Differential Equation (ODE) model which describes the HSL production in the growth media can be written:<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula1.png|250px]]<br />
|}<br />
where:<br />
* [HSL(t)] is the concentration of HSL molecule in the growth medium;<br />
* O.D.600(t) is the growth curve of the considered culture;<br />
* N is the Colony Forming Units (CFU) per O.D.600 unit (i.e. CFU in a well = O.D.600 * N)<br />
* K_HSL is the 3OC6-HSL synthesis rate per cell<br />
<br />
<br />
The analytical solution of this system is:<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula2.png|300px]]<br />
|}<br />
K_HSL can be estimated for t=t_bar, corresponding to the trasncription initiation time, as reported below.<br />
<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula3.png|250px]]<br />
|}<br />
where:<br />
* N was estimated by counting TOP10 colonies obtained by plating serial dilutions of cultures with known O.D.600.<br />
* [HSL(t_bar)] was estimated as described in the section [[Data Analysis - minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>]]<br />
* the O.D.600 time series was measured in each experiment, so its time integral can be computed by trapezoidal numerical integration.<br />
<br />
N was estimated and values are reported below:<br />
<br />
{| border='1' align='center'<br />
| || &nbsp;&nbsp;&nbsp;'''LB'''&nbsp;&nbsp;&nbsp; || &nbsp;&nbsp;&nbsp;'''M9'''&nbsp;&nbsp;&nbsp;<br />
|-<br />
| &nbsp; &nbsp; &nbsp; '''N''' &nbsp; &nbsp; &nbsp;|| &nbsp;&nbsp;2,818 * 10^9 &nbsp;&nbsp;||&nbsp;&nbsp; 2,123 * 10^9 &nbsp;&nbsp;<br />
|}<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data Analysis - minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>===<br />
<br />
Cultures were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for BBa_F2620 - Protocol #4|this protocol]].<br />
<br />
Data from three indipendent wells were averaged and blanked as described in "Preliminary Remarks" section.<br />
<br />
Minimum induction required to activate ''lux pR'' was evaluated by a visual inspection of Scell signal<br />
<br />
<br />
[[Image:pv_MinimumInduction.png|330px|thumb|center|Minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>]]<br />
<br />
<br />
<div align='center'><br />
'''HSL(t_bar)''' : 0,04 nM<br />
</div><br />
HSL(t_bar) was evaluated only in M9 medium because it is a minimum autofluorescence growth medium and it provides the most accurate result in estimating a low fluorescence.<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis for RPU evaluation===<br />
<br />
The RPUs are standard units proposed by Kelly J. et al., 2008, in which the relative transcriptional strength of a promoter can be measured using a reference standard.<br />
<br />
RPUs have been computed as:<br />
[[Image:UNIPV_Pavia_RPU_formula.png|400px|center]]<br />
<br />
in which:<br />
* phi is the promoter of interest and J23101 is the reference standard promoter (taken from the Anderson Promoter Collection);<br />
* F is the blanked fluorescence of the culture, computed by subtracting for each time sample the fluorescence value of a negative control (a non-fluorescent culture). In our experiments, the TOP10 cells bearing BBa_B0032 or BBa_B0033 were usually used because they are RBSs and do not have expression systems for reporter genes;<br />
* ASB is the blanked absorbance (O.D.600) of the culture, computed as described in "Preliminary remarks" section. <br />
RPU measurement has the following advantages (under suitable conditions)<br />
* it is proportional to PoPS (Polymerase Per Second), a very important parameter that expresses the transcription rate of a promoter;<br />
* it uses a reference standard and so measurements can be compared between different laboratories. <br />
The hypotheses on which RPU theory is based can be found in Kelly J. et al., 2008, as well as all the mathematical steps. From our point of view, the main hypotheses that have to be satisfied are the following:<br />
* the reporter protein must have a half life higher than the experiment duration (we use GFPmut3 - <partinfo>BBa_E0040</partinfo> -, which has an estimated half life of at least 24 hours, or an engineered RFP - <partinfo>BBa_E1010</partinfo>, for which the half life has not been measured, but is qualitatively comparable with the GFP's);<br />
* strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in J23101 reference standard.<br />
* steady state must be valid, so (dF/dt)/ASB (proportional to the GFP synthesis rate per cell) must be constant.<br />
<br />
In order to compute the RPUs, the Scell signals ((dGFP/dt)/ASB)) of the promoter of interest and of the reference J23101 were averaged in the time interval corresponding to the exponential growth phase. The boundaries of exponential phase were identified with a visual inspection of the linear phase of the logarithmic growth curve.<br />
<br />
<br><br />
----<br />
<br><br />
<br />
</td></tr></table><br />
<br />
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[[Team:UNIPV-Pavia/Parts/Characterization/NewParts |New Parts]]<hr><br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
</td></tr><br />
<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
</td></tr><br />
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[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]<br><br><br />
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<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts |Improved Parts]]<hr><br />
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<tr align="right"><td align="right" style="padding:10px"><br />
[[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry |Existing Parts from the Registry]]<hr><br />
</td></tr></table> --></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistryTeam:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry2010-10-27T17:13:14Z<p>Susanna: /* BBa_J61001 - Origin of replication */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><td valign=top width="70%"><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<html><p align="center"><font size="4"><b>EXISTING PARTS FROM THE REGISTRY</b></font></p></html><hr><br />
<br />
<tr><td width="100%"><br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><br />
<td align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization]<br />
</td><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th><br />
</tr><br />
</table><br><br />
<br />
=Existing Parts from the Registry: list=<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - R6K Origin of replication|BBa_J61001 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoter collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoter collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - chloramphenicol resistance cassette|BBa_P1004 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - chloramphenicol resistance cassette|BBa_K125500 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=<partinfo>BBa_K300009</partinfo>/<partinfo>BBa_I4102</partinfo> - PoPS->3OC6HSL sender device=<br />
=<partinfo>BBa_F2620</partinfo> - 3OC6HSL -> PoPS Receiver=<br />
=<partinfo>BBa_J61001</partinfo> - R6K Origin of replication=<br />
<br />
=<partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> - constitutive Anderson promoter collection=<br />
=<partinfo>BBa_P1004</partinfo> - chloramphenicol resistance cassette=<br />
<br />
=<partinfo>BBa_K125500</partinfo> - chloramphenicol resistance cassette=<br />
=<partinfo>BBa_J72008</partinfo> - phi80 integration helper plasmid pInt80-649=<br />
<br />
<tr><td><br></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistryTeam:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry2010-10-27T17:12:43Z<p>Susanna: /* Existing Parts from the Registry: list */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><td valign=top width="70%"><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<html><p align="center"><font size="4"><b>EXISTING PARTS FROM THE REGISTRY</b></font></p></html><hr><br />
<br />
<tr><td width="100%"><br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><br />
<td align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization]<br />
</td><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th><br />
</tr><br />
</table><br><br />
<br />
=Existing Parts from the Registry: list=<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - R6K Origin of replication|BBa_J61001 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoter collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoter collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - chloramphenicol resistance cassette|BBa_P1004 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - chloramphenicol resistance cassette|BBa_K125500 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=<partinfo>BBa_K300009</partinfo>/<partinfo>BBa_I4102</partinfo> - PoPS->3OC6HSL sender device=<br />
=<partinfo>BBa_F2620</partinfo> - 3OC6HSL -> PoPS Receiver=<br />
=<partinfo>BBa_J61001</partinfo> - Origin of replication=<br />
=<partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> - constitutive Anderson promoter collection=<br />
=<partinfo>BBa_P1004</partinfo> - chloramphenicol resistance cassette=<br />
<br />
=<partinfo>BBa_K125500</partinfo> - chloramphenicol resistance cassette=<br />
=<partinfo>BBa_J72008</partinfo> - phi80 integration helper plasmid pInt80-649=<br />
<br />
<tr><td><br></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistryTeam:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry2010-10-27T17:11:52Z<p>Susanna: /* BBa_P1004 - R6K Origin of replication */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><td valign=top width="70%"><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<html><p align="center"><font size="4"><b>EXISTING PARTS FROM THE REGISTRY</b></font></p></html><hr><br />
<br />
<tr><td width="100%"><br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><br />
<td align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization]<br />
</td><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th><br />
</tr><br />
</table><br><br />
<br />
=Existing Parts from the Registry: list=<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - Origin of replication|BBa_J61001 - Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoter collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoter collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - R6K Origin of replication|BBa_P1004 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - chloramphenicol resistance cassette|BBa_K125500 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=<partinfo>BBa_K300009</partinfo>/<partinfo>BBa_I4102</partinfo> - PoPS->3OC6HSL sender device=<br />
=<partinfo>BBa_F2620</partinfo> - 3OC6HSL -> PoPS Receiver=<br />
=<partinfo>BBa_J61001</partinfo> - Origin of replication=<br />
=<partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> - constitutive Anderson promoter collection=<br />
=<partinfo>BBa_P1004</partinfo> - chloramphenicol resistance cassette=<br />
<br />
=<partinfo>BBa_K125500</partinfo> - chloramphenicol resistance cassette=<br />
=<partinfo>BBa_J72008</partinfo> - phi80 integration helper plasmid pInt80-649=<br />
<br />
<tr><td><br></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistryTeam:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry2010-10-27T17:06:52Z<p>Susanna: /* Existing Parts from the Registry: list */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><td valign=top width="70%"><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<html><p align="center"><font size="4"><b>EXISTING PARTS FROM THE REGISTRY</b></font></p></html><hr><br />
<br />
<tr><td width="100%"><br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><br />
<td align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization]<br />
</td><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th><br />
</tr><br />
</table><br><br />
<br />
=Existing Parts from the Registry: list=<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - Origin of replication|BBa_J61001 - Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoter collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoter collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - R6K Origin of replication|BBa_P1004 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - chloramphenicol resistance cassette|BBa_K125500 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=<partinfo>BBa_K300009</partinfo>/<partinfo>BBa_I4102</partinfo> - PoPS->3OC6HSL sender device=<br />
=<partinfo>BBa_F2620</partinfo> - 3OC6HSL -> PoPS Receiver=<br />
=<partinfo>BBa_J61001</partinfo> - Origin of replication=<br />
=<partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> - constitutive Anderson promoter collection=<br />
=<partinfo>BBa_P1004</partinfo> - R6K Origin of replication=<br />
<br />
=<partinfo>BBa_K125500</partinfo> - chloramphenicol resistance cassette=<br />
=<partinfo>BBa_J72008</partinfo> - phi80 integration helper plasmid pInt80-649=<br />
<br />
<tr><td><br></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/CharacterizationTeam:UNIPV-Pavia/Parts/Characterization2010-10-27T17:06:00Z<p>Susanna: /* Existing Parts from the Registry */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign=top><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" valign="top" width="100%"><tr><td align="justify" valign="top" style="padding:20px" width="70%" colspan="2"><br />
<html><p align="center"><font size="5"><b>CHARACTERIZATION</b></font></p></html><hr><br><br />
<br />
=Our Parts=<br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th></tr><br />
<!-- <tr><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
</tr> --><br />
</table><br><br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/NewParts |New Parts]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300000 - BioBrick integrative base vector for E. coli |BBa_K300000 - BioBrick integrative base vector for E. coli]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300001 - BioBrick integrative base vector for S. cerevisiae|BBa_K300001 - BioBrick integrative base vector for S. cerevisiae]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain|BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300010 - PoPS-based self-inducible device|BBa_K300010 - PoPS-based self-inducible device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification|BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification]]<br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts |Improved Parts]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300002 - Phasin (PhaP) - head domain|BBa_K300002 - Phasin (PhaP) - head domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300003 - Phasin (PhaP) - internal domain|BBa_K300003 - Phasin (PhaP) - internal domain]]<br />
<br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry |Existing Parts from the Registry]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - Origin of replication|BBa_J61001 - Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoter collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoter collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - R6K Origin of replication|BBa_P1004 - R6K Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - chloramphenicol resistance cassette|BBa_K125500 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<hr><br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=Growth conditions=<br />
===Microplate reader experiments for self-inducible promoters - Protocol #1===<br />
<br />
* 8 ul of long term storage glycerol stock were inoculated in 1 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
* The grown cultures were then diluted 1:100 in 1 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.<br />
* Cultures were then pelletted (2000 rpm, 10 minutes) in order to eliminate the HSL produced during the growth.<br />
* Supernatants were discarded and the pellets were resuspended in 1 ml of LB or M9 + suitable antibiotic and transferred to a 1.5 ml tube<br />
* These cultures were diluted 1:1000 in 1 ml of LB or M9 + suitable antibiotic and aliquoted in a flat-bottom 96-well microplate in triplicate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects, see https://2009.igem.org/Team:UNIPV-Pavia/Methods_Materials/Evaporation). All the wells were filled with a 200 ul volume.<br />
* The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
** 37°C constant for all the experiment;<br />
** sampling time of 5 minutes;<br />
** fluorescence gain of 50;<br />
** O.D. filter was 600 nm;<br />
** GFP filters were 485nm (ex) / 540nm (em);<br />
** 15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
** Variable experiment duration time (from 3 to 24 hours).<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2===<br />
<br />
*8 ul of long term storage glycerol stock were inoculated in 5 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
*The grown cultures were then diluted 1:100 in 5 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.<br />
*These new cultures were diluted to an O.D.600 of 0.02 (measured with a TECAN F200 microplate reader on a 200 ul of volume per well; it is not the 1 cm pathlength cuvette) in 2 ml LB or M9 + suitable antibiotic. In order to have the cultures at the desired O.D.600, the following dilution was performed:<br />
[[Image:UNIPV_Pavia_OD600_dil.png|500px|center]]<br />
*These new dilutions were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects in the frame). All the wells were filled with a 200 ul volume.<br />
*The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
**37°C constant for all the experiment;<br />
**sampling time of 5 minutes;<br />
**fluorescence gain of 50 or 70;<br />
**O.D. filter was 600 nm;<br />
**GFP filters were 485nm (ex) / 540nm (em);<br />
**RFP filters were 535nm (ex) / 620nm (em);<br />
**15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
**Experiment duration time: about 6 hours.<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for 3OC6-HSL quantification by means of <partinfo>BBa_T9002</partinfo> biosensor - Protocol #3===<br />
<br />
The aim of this experiment is the quantification of the 3OC6-HSL produced by cultures harbouring plasmids derived from <partinfo>BBa_K300009</partinfo>. These plasmids are BBa_J231xx-<partinfo>BBa_K300009</partinfo> assemblies, where BBa_J231xx are Anderson Promoter Collection library members. This assembled parts are 3OC6-HSL generators and one important parameter to be evaluated is the concentration of 3OC6-HSL produced.<br />
<br />
The "3OC6-HSL generator" culture was processed as follows:<br />
*<em>Preparation of 3OC6-HSL generating cultures:</em><br />
**8 ul of BBa_J231xx-<partinfo>BBa_K300009</partinfo> long term glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours. <br />
**The grown cultures were then diluted 1:100 in 5 ml of LB supplemented medium and incubated in the same conditions as before for 6 hours.<br />
**O.D.600 was measured, to verify that the growths were synchronized and, if not, final O.D.600 was considered as a correction factor to normalize the amount of 3OC6-HSL produced per cell.<br />
**When all the cultures reached the saturation phase, each falcon was pelletted (2000 rpm, 10 minutes) and supernatants were collected and filtered 0.2 um, in order to eliminate bacterial residues. These supernatants contained the 3OC6-HSL at the concentration produced by cultures. These supernatants were used to "induce" <partinfo>BBa_T9002</partinfo> cultures, in order to quantify the [HSL]. They were conserved at -20°C until the next day.<br />
*<em>Growth and preparation of the biosensor culture of <partinfo>BBa_T9002</partinfo></em><br />
**8 ul of <partinfo>BBa_T9002</partinfo> long term glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
**The grown cultures were then diluted 1:100 in 5 ml of LB supplemented medium and incubated in the same conditions as before until they reached an O.D.600 of 0.07 (measured by Tecan INFINITE F200).<br />
**the required number (see below) of 200 ul aliquots of culture were transferred in each well of a 96-well microplate.<br />
*<em>Calibration curve</em><br />
**A calibration curve was obtained, by inducing in triplicate wells of <partinfo>BBa_T9002</partinfo> with different known HSL concentrations:<br />
***10 uM<br />
***1 uM<br />
***100 nM<br />
*** 50 nM<br />
*** 10 nM<br />
*** 5 nM<br />
***2 nM<br />
***1 nM<br />
*** 0,5 nM<br />
*** 0,1 nM<br />
*** 0 M<br />
**All inductions were performed adding 2ul of "inducer solution" (3OC6-HSL, Sigma Aldrich) at a proper concentration in 200 ul of culture.<br />
*<em>HSL quantification</em><br />
**2ul of supernatants prepared as described above were used to induce 200 ul of <partinfo>BBa_T9002</partinfo> cultures in triplicate. <br />
**If the amount of 3OC6-HSL present in the supernatant was not enough concentrated to trigger the induction of <partinfo>BBa_T9002</partinfo>, a bigger amount of supernatant (X ul) was used to induce the cultures (200-X ul of cultures). In this way, the calibration curve was obtained by using the same amount of <partinfo>BBa_T9002</partinfo> culture (200-X ul).<br />
**Fluorescence and absorbance were measured after 30 min from the induction with a Tecan Infinite F200 microplate reader and, using the information provided by the calibration curve, the amount of 3OC6-HSL present in every supernatant was evaluated (each reported fluorescence value was corrected with the O.D.600 of the culture).<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
<br />
===Microplate reader experiments for <partinfo>BBa_F2620</partinfo> - Protocol #4===<br />
<br />
This protocol is identical to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|this one]], but in addition, after cultures were transferred in the 96-well microplate, 2ul of inducer solution at the proper concentration were added to each well, thus obtaining the final desired concentration.<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=Data Analysis=<br />
<br><br />
===Preliminary remarks===<br />
*All our growth curves have been obtained subtracting for each time sample the broth O.D.600 measurement from that of the culture; broth was considered in the same conditions of the culture (e.g. induced with the same inducer concentration and supplemented with the same antibiotic of the culture).<br />
<br />
*Fluorescence signals have been obtained subtracting for each time sample the fluorescent measurement of a non-fluorescent culture from that of the target culture. The non-fluorescent culture was considered in the same conditions of the target (e.g. induced with the same inducer concentration and with the BioBrick carried by the same plasmid, not encoding a fluorescent protein). This operation allows the removal, from the target fluorescent signal, of the "self-fluorescent" component and the fluorescence signal obtained is "blanked".<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Doubling time evaluation===<br />
The natural logarithm of the growth curves (processed according to the above section) was computed and the linear phase (corresponding to the bacterial exponential growth phase) was isolated by visual inspection. Then the linear regression was performed in order to estimate the slope of the line m. Finally the doubling time was estimated as d=ln(2)/m [minutes].<br />
<br />
In the case of multiple growth curves for a strain, the mean value of the processed curves was computed for each time sample before applying the above described procedure. <br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis for self-inducible promoters (initiation-treshold determination)===<br />
<br />
The task of this analysis is the evaluation of the initiation transcription point (in terms of absorbance) for self-inducible devices. The transition O.D.600 value is named, from now on, O.D.start.<br />
Data from three indipendent wells were averaged and blanked. O.D.600 signals were blanked as described in "Preliminary Remarks" section, while the fluorescence signal was blanked with the fluorescence of <partinfo>BBa_T9002</partinfo> part, assembled in the same plasmid of the considered promoter. This operation allows the removal from the fluorescence signal of the autofluorescence component and of the “leaky” component (due to the leakage of ''lux pR'' promoter in absence of the autoinducer molecule HSL).<br />
<br />
O.D.start was evaluated by computing the ''Scell'' (GFPmut3 synthesis rate per cell) signal for the desired self-inducible promoter.<br />
<br />
Scell was obtained by computing (1/O.D.600)*dGFP/dt, where O.D.600 and GFP are the normalized absorbance and fluorescence signals.<br />
<br />
The goal is the estimation of the critical O.D.600 value (O.D.start) at which the Scell significantly increases. Because Scell is a very noisy signal, a threshold value which takes into account the noise variability was proposed and it is described below.<br />
<br />
Two different signals, measured from independent samples of the same non-fluorescent culture in the same experiment, are considered: C1 and C2. The fluorescence signal of C1 and C2 (F_C1 and F_C2 respectively) can be thought as the addition of a “real signal” and a noise component.<br />
<br />
[[Image:UNIPV_Pavia_noise1.png|400px|center]]<br />
<br />
F_C1 and F_C2 have the same expected value and the same standard deviation, since they are two independent realizations of the same aleatory process: in fact, they are two time series acquired from the same cultures in the same growth conditions by the same instrument.<br />
Noise signal was computed as:<br />
<br />
[[Image:UNIPV_Pavia_noise2.png|150px|center]]<br />
<br />
An interesting signal is the Scell of ''N'' time series. It can be evaluated as the time derivative of ''N'', divided by O.D.600 of the culture. It has the behaviour shown in figure:<br />
<br />
[[Image:UNIPV_Pavia_rumoreNoiseScell.png|600px|center|Scell Noise]]<br />
It is fair to say that the noise of Scell is bigger for low O.D.600 values and its amplitude decreases dramatically for higher O.D.600 values (e.g.: O.D.600>0.1 measured with the TECAN Infinite F200 in a 96-well microplate).<br />
<br />
The O.D.600-dependent noise model proposed for the Scell signal is:<br />
<br />
[[Image:UNIPV_Pavia_ModelNoiseScell.png|300px|center|Scell noise model]]<br />
<br />
According to the formula reported above, it is possible to obtain the sigma_bar value as follows:<br />
#multiply the Scell time series values with the corresponding O.D.600;<br />
#the sigma_bar constant value can be estimated as the standard deviation of this signal.<br />
<br />
The Scell*O.D.600 signal behaviour is reported below:<br />
<br />
[[Image:UNIPV_Pavia_rumoreNoise.png|600px|center|Measurement Noise]]<br />
<br />
As this figure shows, the noise amplitude of this signal is no more O.D.600-dependent and the sigma_bar can be obtained.<br />
<br />
<br />
The evaluation of the O.D.start can be performed supposing that the transcription starts when the signal is significantly different from the noise. A threshold on the signal amplitude was estabilished as reported here:<br />
<br />
[[Image:UNIPV_Pavia_threshold.png|300px|center|variance threshold]]<br />
<br />
In order to make the method more robust, induction was considered only when 5 consecutive values exceeded the threshold amplitude.<br />
<br />
<br />
The operation of subtracting the two signals F_C1 and F_C2 is analogous to the “blanking” operation performed on data, as described previously. For, this reason, under the hypothesis that the signals have the same variance (this is a consistent hypothesis, since they are measured by the same instrument in the same experimental conditions), this argument can be extended to the “blanked” data considered in the processing. <br />
In particular, it is evident that the "blanked" signal is affected by the same noise described above and thus, the noise of Scell signal is analogous to the one described before.<br />
Thus, this threshold was used to compute the O.D.start for the cultures. This heuristic algorithm was implemented in Matlab and analysis results are reported in the “results” section.<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data Analysis to estimate the HSL synthesis rate per cell===<br />
<br />
<br />
The autoinducer synthesys rate per cell is a very important parameter to be evaluated in quorum sensing systems. <br />
In fact the knowledge of this parameter enables the rational design of cell-communication systems, such as the self inducible devices studied in this project.<br />
<br />
A model based approach was proposed to estimate this interesting parameter. <br />
<br />
Under the hypotheses that:<br />
* no HSL is present at the beginning of the experiment - consistent hypothesis because a growth medium washing step is always performed before the experiment start in order to remove the HSL produced until that moment,<br />
* the HSL synthesis rate is not time-dependent (cells produce HSL at the same rate in each growth phase - this hypotesis simplifies the data analysis, but it should be further validated),<br />
* the half life of the autoinducer is much longer than the experiment duration (i.e. the degradation of the molecule is negligible - this hypothesis is consistent, since no HSL-degrading enzyme is present in ''E. coli''),<br />
<br />
an Ordinary Differential Equation (ODE) model which describes the HSL production in the growth media can be written:<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula1.png|250px]]<br />
|}<br />
where:<br />
* [HSL(t)] is the concentration of HSL molecule in the growth medium;<br />
* O.D.600(t) is the growth curve of the considered culture;<br />
* N is the Colony Forming Units (CFU) per O.D.600 unit (i.e. CFU in a well = O.D.600 * N)<br />
* K_HSL is the 3OC6-HSL synthesis rate per cell<br />
<br />
<br />
The analytical solution of this system is:<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula2.png|300px]]<br />
|}<br />
K_HSL can be estimated for t=t_bar, corresponding to the trasncription initiation time, as reported below.<br />
<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula3.png|250px]]<br />
|}<br />
where:<br />
* N was estimated by counting TOP10 colonies obtained by plating serial dilutions of cultures with known O.D.600.<br />
* [HSL(t_bar)] was estimated as described in the section [[Data Analysis - minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>]]<br />
* the O.D.600 time series was measured in each experiment, so its time integral can be computed by trapezoidal numerical integration.<br />
<br />
N was estimated and values are reported below:<br />
<br />
{| border='1' align='center'<br />
| || &nbsp;&nbsp;&nbsp;'''LB'''&nbsp;&nbsp;&nbsp; || &nbsp;&nbsp;&nbsp;'''M9'''&nbsp;&nbsp;&nbsp;<br />
|-<br />
| &nbsp; &nbsp; &nbsp; '''N''' &nbsp; &nbsp; &nbsp;|| &nbsp;&nbsp;2,818 * 10^9 &nbsp;&nbsp;||&nbsp;&nbsp; 2,123 * 10^9 &nbsp;&nbsp;<br />
|}<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data Analysis - minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>===<br />
<br />
Cultures were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for BBa_F2620 - Protocol #4|this protocol]].<br />
<br />
Data from three indipendent wells were averaged and blanked as described in "Preliminary Remarks" section.<br />
<br />
Minimum induction required to activate ''lux pR'' was evaluated by a visual inspection of Scell signal<br />
<br />
<br />
[[Image:pv_MinimumInduction.png|330px|thumb|center|Minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>]]<br />
<br />
<br />
<div align='center'><br />
'''HSL(t_bar)''' : 0,04 nM<br />
</div><br />
HSL(t_bar) was evaluated only in M9 medium because it is a minimum autofluorescence growth medium and it provides the most accurate result in estimating a low fluorescence.<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis for RPU evaluation===<br />
<br />
The RPUs are standard units proposed by Kelly J. et al., 2008, in which the relative transcriptional strength of a promoter can be measured using a reference standard.<br />
<br />
RPUs have been computed as:<br />
[[Image:UNIPV_Pavia_RPU_formula.png|400px|center]]<br />
<br />
in which:<br />
* phi is the promoter of interest and J23101 is the reference standard promoter (taken from the Anderson Promoter Collection);<br />
* F is the blanked fluorescence of the culture, computed by subtracting for each time sample the fluorescence value of a negative control (a non-fluorescent culture). In our experiments, the TOP10 cells bearing BBa_B0032 or BBa_B0033 were usually used because they are RBSs and do not have expression systems for reporter genes;<br />
* ASB is the blanked absorbance (O.D.600) of the culture, computed as described in "Preliminary remarks" section. <br />
RPU measurement has the following advantages (under suitable conditions)<br />
* it is proportional to PoPS (Polymerase Per Second), a very important parameter that expresses the transcription rate of a promoter;<br />
* it uses a reference standard and so measurements can be compared between different laboratories. <br />
The hypotheses on which RPU theory is based can be found in Kelly J. et al., 2008, as well as all the mathematical steps. From our point of view, the main hypotheses that have to be satisfied are the following:<br />
* the reporter protein must have a half life higher than the experiment duration (we use GFPmut3 - <partinfo>BBa_E0040</partinfo> -, which has an estimated half life of at least 24 hours, or an engineered RFP - <partinfo>BBa_E1010</partinfo>, for which the half life has not been measured, but is qualitatively comparable with the GFP's);<br />
* strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in J23101 reference standard.<br />
* steady state must be valid, so (dF/dt)/ASB (proportional to the GFP synthesis rate per cell) must be constant.<br />
<br />
In order to compute the RPUs, the Scell signals ((dGFP/dt)/ASB)) of the promoter of interest and of the reference J23101 were averaged in the time interval corresponding to the exponential growth phase. The boundaries of exponential phase were identified with a visual inspection of the linear phase of the logarithmic growth curve.<br />
<br />
<br><br />
----<br />
<br><br />
<br />
</td></tr></table><br />
<br />
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[[Team:UNIPV-Pavia/Parts/Characterization/NewParts |New Parts]]<hr><br />
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[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
</td></tr><br />
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[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
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[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]<br><br><br />
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[[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts |Improved Parts]]<hr><br />
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[[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry |Existing Parts from the Registry]]<hr><br />
</td></tr></table> --></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistryTeam:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry2010-10-27T17:05:32Z<p>Susanna: /* BBa_P1004 - Origin of replication */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><td valign=top width="70%"><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<html><p align="center"><font size="4"><b>EXISTING PARTS FROM THE REGISTRY</b></font></p></html><hr><br />
<br />
<tr><td width="100%"><br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><br />
<td align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization]<br />
</td><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="25%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th><br />
</tr><br />
</table><br><br />
<br />
=Existing Parts from the Registry: list=<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - Origin of replication|BBa_J61001 - Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoter collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoter collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - Origin of replication|BBa_P1004 - Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - chloramphenicol resistance cassette|BBa_K125500 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=<partinfo>BBa_K300009</partinfo>/<partinfo>BBa_I4102</partinfo> - PoPS->3OC6HSL sender device=<br />
=<partinfo>BBa_F2620</partinfo> - 3OC6HSL -> PoPS Receiver=<br />
=<partinfo>BBa_J61001</partinfo> - Origin of replication=<br />
=<partinfo>BBa_J23100</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23118</partinfo> - constitutive Anderson promoter collection=<br />
=<partinfo>BBa_P1004</partinfo> - R6K Origin of replication=<br />
<br />
=<partinfo>BBa_K125500</partinfo> - chloramphenicol resistance cassette=<br />
=<partinfo>BBa_J72008</partinfo> - phi80 integration helper plasmid pInt80-649=<br />
<br />
<tr><td><br></div>Susannahttp://2010.igem.org/File:Pv_RPUvsOD_HCLC_M9.pngFile:Pv RPUvsOD HCLC M9.png2010-10-27T16:57:34Z<p>Susanna: uploaded a new version of "Image:Pv RPUvsOD HCLC M9.png"</p>
<hr />
<div></div>Susannahttp://2010.igem.org/File:Pv_RPUvsOD_HCHC_M9.pngFile:Pv RPUvsOD HCHC M9.png2010-10-27T16:57:29Z<p>Susanna: uploaded a new version of "Image:Pv RPUvsOD HCHC M9.png"</p>
<hr />
<div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAutoTeam:UNIPV-Pavia/Project/PromotoriAuto2010-10-27T15:26:05Z<p>Susanna: /* Modulation of plasmid copy number */</p>
<hr />
<div>__NOTOC__<br />
{{UNIPV-Pavia/header}} <br />
<br />
<br />
<table width="100%" border="0" align="center"><br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td align="justify" valign="top" style="padding:20px"><br />
<p align="center" valign="top"><br><br />
<font size="5" face="Cataneo BT" align="center" valign="top">'''''ProteInProgress: a cellular assembly line for protein manufacturing'''''</font><br />
</p><br />
<hr><br />
<br><br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/motivation"><br />
<img src="https://static.igem.org/mediawiki/2010/b/be/UNIPV_Pavia_motivation2_BN.jpg" width="75px" height="75px" alt="Motivation" title="Motivation"/><br />
</a><br />
</html></td><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/solution"><br />
<img src="https://static.igem.org/mediawiki/2010/c/c8/UNIPV_Pavia_solution2_BN.jpg" width="75px" height="75px" alt="Solutions" title="Solutions"/></a><br />
</html></td><br />
<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/results"><br />
<img src="https://static.igem.org/mediawiki/2010/7/77/UNIPV_Pavia_result.jpg" width="75px" height="75px" alt="Implementation & Results" title="Implementation & Results"/><br />
</a><br />
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<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/references"><br />
<img src="https://static.igem.org/mediawiki/2010/6/61/UNIPV_Paviareferences_BN.jpg" width="75px" height="75px" alt="References" title="References"/></a><br />
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</table><br />
<br />
<html><a name="indice"/></a></html><br />
<br />
<p align="center" valign="top"><br><br />
<font size="5" align="center" valign="top"><b>Implementation and Results: Self-Inducible promoters</b></font><br />
</p><br />
<br><br />
<hr><br />
<br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAuto"><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/PromotoriAuto|Self-inducible promoters]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntColi"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/UNIPV_Pavia_Icona_intColi.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntColi|Integrative standard vector for E. coli]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntYeast"><br />
<img src="https://static.igem.org/mediawiki/2010/0/03/UNIPV_Pavia_Icona_IntYeast.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntYeast|Integrative standard vector for yeast]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/FasinaInteina"><br />
<img src="https://static.igem.org/mediawiki/2010/7/74/UNIPV_Pavia_Icona_Bioplastica.GIF" width="75px" height="75px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/FasinaInteina|Purification of proteins]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/><br />
</html><br />
<br />
=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein production</b>===<br />
<br />
'''Experimental implementation:''' <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:<br />
<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>Strength (a.u.)<br>reported in the Registry</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 2547<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||1791<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||623<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||1185<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||844<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||256<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||396<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||1429<br />
|}<br />
<br />
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]], using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:<br />
*high copy number plasmids and LB;<br />
*high copy number plasmids and M9;<br />
*low copy number plasmids and M9.<br />
It was not possible to evaluate promoters activities in low copy number plasmids in LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2]]. Data were analyzed as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]]; <br />
<br />
'''Results''': results are shown here.<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI fragment of <partinfo>BBa_J61002</partinfo>-BBa_J231xx in <partinfo>pSB4C5</partinfo> vector, in order to transfer the promoter and the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
<br />
Doubling times were evaluated for the described cultures (HC stands for High Copy and LC stands for Low Copy):<br />
{| align='center' border='1'<br />
|rowspan='2'|<b>Promoter</b><br />
|colspan='3'| <b>doubling time [minutes]</b><br />
|-<br />
| LB in HC plasmid || M9 in HC plasmid || M9 in LC plasmid<br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 33.75 <br> ± <br> 1.34 || 82.53 <br> ± <br> 2.45 || 86.11 <br> ± <br> 4.45<br />
|-<br />
|<partinfo>BBa_J23101</partinfo> || 35.93 <br> ± <br> 0.62 || 82.68 <br> ± <br> 1.84 || 86.42 <br> ± <br> 1.91<br />
|-<br />
|<partinfo>BBa_J23105</partinfo> || 29.86 <br> ± <br> 0.33 || 63.09 <br> ± <br> 7.08 || 85.00 <br> ± <br> 5.13<br />
|-<br />
|<partinfo>BBa_J23106</partinfo> || 29.17 <br> ± <br> 0.96 || 68.11 <br> ± <br> 4.25 || 88.71 <br> ± <br> 0.90<br />
|-<br />
|<partinfo>BBa_J23110</partinfo> || 31.28 <br> ± <br> 0.42 || 67.52 <br> ± <br> 5.87 || 76.15 <br> ± <br> 2.16<br />
|-<br />
|<partinfo>BBa_J23114</partinfo> || 28.97 <br> ± <br> 0.49 || 59.44 <br> ± <br> 5.20 || 80.12 <br> ± <br> 0.95<br />
|-<br />
|<partinfo>BBa_J23116</partinfo> || 28.14 <br> ± <br> 0.25 || 72.74 <br> ± <br> 0.37 || 81.68 <br> ± <br> 3.08<br />
|-<br />
|<partinfo>BBa_J23118</partinfo> || 32.84 <br> ± <br> 0.31 || 73.64 <br> ± <br> 2.41 || 89.86 <br> ± <br> 2.93<br />
|}<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the tested conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced amounts of LuxI protein that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformants could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluorescent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced by autoinducer generators</b>===<br />
<br />
'''Experimental implementation:''' The autoinducer generators <partinfo>BBa_K300030</partinfo>, <partinfo>BBa_K300028</partinfo>, <partinfo>BBa_K300029</partinfo>, <partinfo>BBa_K300025</partinfo>, <partinfo>BBa_K300026</partinfo> and <partinfo>BBa_K300027</partinfo> were, thus, characterized by measuring the concentration of HSL released in the medium of cultures grown for 6 hours. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 sensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results:''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table below:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced by autoinducer generators in high copy vector.]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> after a 6 hour cell growth is reported in Fig.9 and in the table below:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced by the autoinducer generators in low copy vector.]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) in high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized in tables.<br />
<br />
<br />
<div align='center>Sender/Receiver devices assembled as a unique BioBrick part on the same vector</div><br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender/Receiver Device Vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|}<br />
<br />
<div align='center>Sender devices assembled on low copy number vector and Receiver device on high copy number vector</div><br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center' width='80%'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|300px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|300px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|300px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|300px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]]<br />
*K_HSL is the HSL synthesys rate per cell, it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis to estimate the HSL synthesys rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double. It was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
<br>[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> ± <br> *<br />
| 8.94 10^-17 <br> ± <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> ± <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> ± <br> *<br />
| 7.53 10^-18 <br> ± <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> ± <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br>[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
<br />
'''Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br></td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300030.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
|Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br></td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300028.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300029</partinfo> (wiki name: I16) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300029.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.31 <br> ± <br> *<br />
| 1.17 10^-16 <br> ± <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> ± <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300025.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300026.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300027.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 70%; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two self-inducible devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were realized and characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell and an algorithm was proposed, in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In figure, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported, both for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid). <br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the Signal Molucule production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by data. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate results stronger at the increase of promoter's strength. <br />
<br />
For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably due to the fact that too high synthesis rate of HSL are injurious for the cell. <br />
A range of RPU values for promoters that control the Sender device was identified. When the PoPs-in signal belongs to this range, the expected behaviour is confirmed by experimental data. <br />
The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices, able to start the production of a target protein at different O.D._start values, in any cellular growth phase.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAutoTeam:UNIPV-Pavia/Project/PromotoriAuto2010-10-27T15:18:07Z<p>Susanna: /* Results */</p>
<hr />
<div>__NOTOC__<br />
{{UNIPV-Pavia/header}} <br />
<br />
<br />
<table width="100%" border="0" align="center"><br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td align="justify" valign="top" style="padding:20px"><br />
<p align="center" valign="top"><br><br />
<font size="5" face="Cataneo BT" align="center" valign="top">'''''ProteInProgress: a cellular assembly line for protein manufacturing'''''</font><br />
</p><br />
<hr><br />
<br><br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/motivation"><br />
<img src="https://static.igem.org/mediawiki/2010/b/be/UNIPV_Pavia_motivation2_BN.jpg" width="75px" height="75px" alt="Motivation" title="Motivation"/><br />
</a><br />
</html></td><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/solution"><br />
<img src="https://static.igem.org/mediawiki/2010/c/c8/UNIPV_Pavia_solution2_BN.jpg" width="75px" height="75px" alt="Solutions" title="Solutions"/></a><br />
</html></td><br />
<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/results"><br />
<img src="https://static.igem.org/mediawiki/2010/7/77/UNIPV_Pavia_result.jpg" width="75px" height="75px" alt="Implementation & Results" title="Implementation & Results"/><br />
</a><br />
</html></td><br />
<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/references"><br />
<img src="https://static.igem.org/mediawiki/2010/6/61/UNIPV_Paviareferences_BN.jpg" width="75px" height="75px" alt="References" title="References"/></a><br />
</html></td><br />
</tr><br />
</table><br />
<br />
<html><a name="indice"/></a></html><br />
<br />
<p align="center" valign="top"><br><br />
<font size="5" align="center" valign="top"><b>Implementation and Results: Self-Inducible promoters</b></font><br />
</p><br />
<br><br />
<hr><br />
<br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAuto"><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/PromotoriAuto|Self-inducible promoters]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntColi"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/UNIPV_Pavia_Icona_intColi.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntColi|Integrative standard vector for E. coli]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntYeast"><br />
<img src="https://static.igem.org/mediawiki/2010/0/03/UNIPV_Pavia_Icona_IntYeast.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntYeast|Integrative standard vector for yeast]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/FasinaInteina"><br />
<img src="https://static.igem.org/mediawiki/2010/7/74/UNIPV_Pavia_Icona_Bioplastica.GIF" width="75px" height="75px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/FasinaInteina|Purification of proteins]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/><br />
</html><br />
<br />
=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein production</b>===<br />
<br />
'''Experimental implementation:''' <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:<br />
<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>Strength (a.u.)<br>reported in the Registry</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 2547<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||1791<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||623<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||1185<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||844<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||256<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||396<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||1429<br />
|}<br />
<br />
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]], using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:<br />
*high copy number plasmids and LB;<br />
*high copy number plasmids and M9;<br />
*low copy number plasmids and M9.<br />
It was not possible to evaluate promoters activities in low copy number plasmids in LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2]]. Data were analyzed as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]]; <br />
<br />
'''Results''': results are shown here.<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI fragment of <partinfo>BBa_J61002</partinfo>-BBa_J231xx in <partinfo>pSB4C5</partinfo> vector, in order to transfer the promoter and the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
<br />
Doubling times were evaluated for the described cultures (HC stands for High Copy and LC stands for Low Copy):<br />
{| align='center' border='1'<br />
|rowspan='2'|<b>Promoter</b><br />
|colspan='3'| <b>doubling time [minutes]</b><br />
|-<br />
| LB in HC plasmid || M9 in HC plasmid || M9 in LC plasmid<br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || || ||<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>|| || ||<br />
|}<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the tested conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced amounts of LuxI protein that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformants could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluorescent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced by autoinducer generators</b>===<br />
<br />
'''Experimental implementation:''' The autoinducer generators <partinfo>BBa_K300030</partinfo>, <partinfo>BBa_K300028</partinfo>, <partinfo>BBa_K300029</partinfo>, <partinfo>BBa_K300025</partinfo>, <partinfo>BBa_K300026</partinfo> and <partinfo>BBa_K300027</partinfo> were, thus, characterized by measuring the concentration of HSL released in the medium of cultures grown for 6 hours. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 sensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results:''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table below:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced by autoinducer generators in high copy vector.]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> after a 6 hour cell growth is reported in Fig.9 and in the table below:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced by the autoinducer generators in low copy vector.]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) in high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized here:<br />
<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan="2" align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center' width='80%'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|300px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|300px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|300px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|300px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]]<br />
*K_HSL is the HSL synthesys rate per cell, it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis to estimate the HSL synthesys rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double. It was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
<br>[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> ± <br> *<br />
| 8.94 10^-17 <br> ± <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> ± <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> ± <br> *<br />
| 7.53 10^-18 <br> ± <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> ± <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br>[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
<br />
'''Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br></td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300030.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
|Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br></td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300028.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300029</partinfo> (wiki name: I16) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300029.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.31 <br> ± <br> *<br />
| 1.17 10^-16 <br> ± <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> ± <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300025.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300026.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<table><tr><td><font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
</td><td><partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo></td></tr><br />
<tr><td>[[Image:pv_BBa_K300027.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 70%; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two self-inducible devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were realized and characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell and an algorithm was proposed, in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In figure, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported, both for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid). <br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the Signal Molucule production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by data. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate results stronger at the increase of promoter's strength. <br />
<br />
For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably due to the fact that too high synthesis rate of HSL are injurious for the cell. <br />
A range of RPU values for promoters that control the Sender device was identified. When the PoPs-in signal belongs to this range, the expected behaviour is confirmed by experimental data. <br />
The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices, able to start the production of a target protein at different O.D._start values, in any cellular growth phase.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAutoTeam:UNIPV-Pavia/Project/PromotoriAuto2010-10-27T14:20:17Z<p>Susanna: /* Results */</p>
<hr />
<div>__NOTOC__<br />
{{UNIPV-Pavia/header}} <br />
<br />
<br />
<table width="100%" border="0" align="center"><br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td align="justify" valign="top" style="padding:20px"><br />
<p align="center" valign="top"><br><br />
<font size="5" face="Cataneo BT" align="center" valign="top">'''''ProteInProgress: a cellular assembly line for protein manufacturing'''''</font><br />
</p><br />
<hr><br />
<br><br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/motivation"><br />
<img src="https://static.igem.org/mediawiki/2010/b/be/UNIPV_Pavia_motivation2_BN.jpg" width="75px" height="75px" alt="Motivation" title="Motivation"/><br />
</a><br />
</html></td><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/solution"><br />
<img src="https://static.igem.org/mediawiki/2010/c/c8/UNIPV_Pavia_solution2_BN.jpg" width="75px" height="75px" alt="Solutions" title="Solutions"/></a><br />
</html></td><br />
<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/results"><br />
<img src="https://static.igem.org/mediawiki/2010/7/77/UNIPV_Pavia_result.jpg" width="75px" height="75px" alt="Implementation & Results" title="Implementation & Results"/><br />
</a><br />
</html></td><br />
<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/references"><br />
<img src="https://static.igem.org/mediawiki/2010/6/61/UNIPV_Paviareferences_BN.jpg" width="75px" height="75px" alt="References" title="References"/></a><br />
</html></td><br />
</tr><br />
</table><br />
<br />
<html><a name="indice"/></a></html><br />
<br />
<p align="center" valign="top"><br><br />
<font size="5" align="center" valign="top"><b>Implementation and Results: Self-Inducible promoters</b></font><br />
</p><br />
<br><br />
<hr><br />
<br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAuto"><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/PromotoriAuto|Self-inducible promoters]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntColi"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/UNIPV_Pavia_Icona_intColi.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntColi|Integrative standard vector for E. coli]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntYeast"><br />
<img src="https://static.igem.org/mediawiki/2010/0/03/UNIPV_Pavia_Icona_IntYeast.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntYeast|Integrative standard vector for yeast]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/FasinaInteina"><br />
<img src="https://static.igem.org/mediawiki/2010/7/74/UNIPV_Pavia_Icona_Bioplastica.GIF" width="75px" height="75px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/FasinaInteina|Purification of proteins]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/><br />
</html><br />
<br />
=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein production</b>===<br />
<br />
'''Experimental implementation:''' <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:<br />
<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>Strength (a.u.)<br>reported in the Registry</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 2547<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||1791<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||623<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||1185<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||844<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||256<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||396<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||1429<br />
|}<br />
<br />
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]], using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:<br />
*high copy number plasmids and LB;<br />
*high copy number plasmids and M9;<br />
*low copy number plasmids and M9.<br />
It was not possible to evaluate promoters activities in low copy number plasmids in LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2]] and data were analyzed as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]]; <br />
<br />
'''Results''': results are shown here.<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI the <partinfo>BBa_J61002</partinfo>-BBa_J231xx EcoRI-PstI fragment in <partinfo>pSB4C5</partinfo>, in order to transfer the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
<br />
Doubling times were evaluated for the described cultures:<br />
{| align='center' border='1'<br />
|rowspan='2'|<b>Promoter</b><br />
|colspan='3'| <b>doubling time [minutes]</b><br />
|-<br />
| LB in HC plasmid || M9 in HC plasmid || M9 in LC plasmid<br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || || ||<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>|| || ||<br />
|}<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced LuxI protein amounts that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformans could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluoresent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced</b>===<br />
<br />
'''Experimental implementation''' The new parts were, thus, characterized, measuring the HSL concentration released in the medium after a 6 hour growth of the cultures. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 sensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts contained in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in high copy plasmid]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced after 6 hours growth by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> is reported in Fig.9 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in low copy plasmid]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) in high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized here:<br />
<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan="2" align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center' width='80%'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|300px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|300px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|300px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|300px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]]<br />
*K_HSL is the HSL synthesys rate per cell, it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis to estimate the HSL synthesys rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double. It was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
<br>[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> ± <br> *<br />
| 8.94 10^-17 <br> ± <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> ± <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> ± <br> *<br />
| 7.53 10^-18 <br> ± <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> ± <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br>[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
<br />
'''Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br><br />
<table><tr><td>[[Image:pv_BBa_K300030.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
|Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br><br />
<table><tr><td>[[Image:pv_BBa_K300028.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300029</partinfo> (wiki name: I16) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br><br />
<table><tr><td>[[Image:pv_BBa_K300029.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.31 <br> ± <br> *<br />
| 1.17 10^-16 <br> ± <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> ± <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br><br />
<table><tr><td>[[Image:pv_BBa_K300025.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br><br />
<table><tr><td>[[Image:pv_BBa_K300026.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br><br />
<table><tr><td>[[Image:pv_BBa_K300027.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 70%; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two self-inducible devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were realized and characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell and an algorithm was proposed, in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In figure, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported, both for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid). <br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the Signal Molucule production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by data. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate results stronger at the increase of promoter's strength. <br />
<br />
For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably due to the fact that too high synthesis rate of HSL are injurious for the cell. <br />
A range of RPU values for promoters that control the Sender device was identified. When the PoPs-in signal belongs to this range, the expected behaviour is confirmed by experimental data. <br />
The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices, able to start the production of a target protein at different O.D._start values, in any cellular growth phase.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/CharacterizationTeam:UNIPV-Pavia/Parts/Characterization2010-10-27T14:15:36Z<p>Susanna: /* Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2 */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign=top><br />
<br />
<!-- Contenuti --><br />
{{UNIPV-Pavia/Style}}<br />
<table border="0" align="center" valign="top" width="100%"><tr><td align="justify" valign="top" style="padding:20px" width="70%" colspan="2"><br />
<html><p align="center"><font size="5"><b>CHARACTERIZATION</b></font></p></html><hr><br><br />
<br />
=Our Parts=<br />
<table class="cont" border="2" width="100%" align="center"><br />
<tr><th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts]<br />
</th><br />
<th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts]<br />
</th><br />
<th align="center" width="33%"><br />
[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
</th></tr><br />
<!-- <tr><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
<td>[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300010 |BBa_K300010]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300004 |BBa_K300004]]<br />
[[Team:UNIPV-Pavia/Parts/Characterization/NewParts #BBa_K300000 |BBa_K300000]]</td><br />
</tr> --><br />
</table><br><br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/NewParts |New Parts]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300000 - BioBrick integrative base vector for E. coli |BBa_K300000 - BioBrick integrative base vector for E. coli]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300001 - BioBrick integrative base vector for S. cerevisiae|BBa_K300001 - BioBrick integrative base vector for S. cerevisiae]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain|BBa_K300004 - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300010 - PoPS-based self-inducible device|BBa_K300010 - PoPS-based self-inducible device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/NewParts#BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification|BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095 and BBa_K300084 - Phasin and Intein-based tags for protein purification]]<br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts |Improved Parts]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300002 - Phasin (PhaP) - head domain|BBa_K300002 - Phasin (PhaP) - head domain]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300003 - Phasin (PhaP) - internal domain|BBa_K300003 - Phasin (PhaP) - internal domain]]<br />
<br />
<br />
==[[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry |Existing Parts from the Registry]]==<br />
<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device|BBa_K300009/BBa_I4102 - PoPS->3OC6HSL sender device]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_F2620 - 3OC6HSL -> PoPS Receiver|BBa_F2620 - 3OC6HSL -> PoPS Receiver]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J61001 - Origin of replication|BBa_J61001 - Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoter collection|BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 - constitutive Anderson promoter collection]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_P1004 - Origin of replication|BBa_P1004 - Origin of replication]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_K125500 - chloramphenicol resistance cassette|BBa_K125500 - chloramphenicol resistance cassette]]<br />
* [[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry#BBa_J72008 - phi80 integration helper plasmid pInt80-649|BBa_J72008 - phi80 integration helper plasmid pInt80-649]]<br />
<hr><br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=Growth conditions=<br />
===Microplate reader experiments for self-inducible promoters - Protocol #1===<br />
<br />
* 8 ul of long term storage glycerol stock were inoculated in 1 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
* The grown cultures were then diluted 1:100 in 1 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.<br />
* These new cultures were then pelletted (2000 rpm, 10 minutes) in order to eliminate the HSL produced during the growth.<br />
* Supernatants were discarded and the pellets were resuspended in 1 ml of LB or M9 + suitable antibiotic and transferred to a 1.5 ml tube<br />
* These cultures were diluted 1:1000 in 1 ml of LB or M9 + suitable antibiotic and aliquoted in a flat-bottom 96-well microplate in triplicate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects in the frame). All the wells were filled with a 200 ul volume.<br />
* The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
** 37°C constant for all the experiment;<br />
** sampling time of 5 minutes;<br />
** fluorescence gain of 50;<br />
** O.D. filter was 600 nm;<br />
** GFP filters were 485nm (ex) / 540nm (em);<br />
** 15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
** Variable experiment duration time (from 3 to 24 hours).<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2===<br />
<br />
*8 ul of long term storage glycerol stock were inoculated in 5 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
*The grown cultures were then diluted 1:100 in 5 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.<br />
*These new cultures were diluted to an O.D.600 of 0.02 (measured with a TECAN F200 microplate reader on a 200 ul of volume per well; it is not the 1 cm pathlength cuvette) in 2 ml LB or M9 + suitable antibiotic. In order to have the cultures at the desired O.D.600, the following dilution was performed:<br />
[[Image:UNIPV_Pavia_OD600_dil.png|500px|center]]<br />
*These new dilutions were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects in the frame). All the wells were filled with a 200 ul volume.<br />
*The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:<br />
**37°C constant for all the experiment;<br />
**sampling time of 5 minutes;<br />
**fluorescence gain of 50 or 70;<br />
**O.D. filter was 600 nm;<br />
**GFP filters were 485nm (ex) / 540nm (em);<br />
**RFP filters were 535nm (ex) / 620nm (em);<br />
**15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.<br />
**Experiment duration time: about 6 hours.<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Microplate reader experiments for 3OC6-HSL quantification by means of <partinfo>BBa_T9002</partinfo> biosensor - Protocol #3===<br />
<br />
The aim of this experiment is the quantification of the 3OC6-HSL produced by cultures harbouring plasmids derived from <partinfo>BBa_K300009</partinfo>. These plasmids are BBa_J231xx-<partinfo>BBa_K300009</partinfo> assemblies, where BBa_J231xx are Anderson Promoter Collection library members. This assembled parts are 3OC6-HSL generators and one important parameter to be evaluated is the concentration of 3OC6-HSL produced.<br />
<br />
The "3OC6-HSL generator" culture was processed as follows:<br />
*<em>Preparation of 3OC6-HSL generating cultures:</em><br />
**8 ul of BBa_J231xx-<partinfo>BBa_K300009</partinfo> long term glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours. <br />
**The grown cultures were then diluted 1:100 in 5 ml of LB supplemented medium and incubated in the same conditions as before for 6 hours.<br />
**O.D.600 was measured, to verify that the growths were synchronized and, if not, final O.D.600 was considered as a correction factor to normalize the amount of 3OC6-HSL produced per cell.<br />
**When all the cultures reached the saturation phase, each falcon was pelletted (2000 rpm, 10 minutes) and supernatants were collected and filtered 0.2 um, in order to eliminate bacterial residues. These supernatants contained the 3OC6-HSL at the concentration produced by cultures. These supernatants were used to "induce" <partinfo>BBa_T9002</partinfo> cultures, in order to quantify the [HSL]. They were conserved at -20°C until the next day.<br />
*<em>Growth and preparation of the biosensor culture of <partinfo>BBa_T9002</partinfo></em><br />
**8 ul of <partinfo>BBa_T9002</partinfo> long term glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.<br />
**The grown cultures were then diluted 1:100 in 5 ml of LB supplemented medium and incubated in the same conditions as before until they reached an O.D.600 of 0.07 (measured by Tecan INFINITE F200).<br />
**the required number (see below) of 200 ul aliquots of culture were transferred in each well of a 96-well microplate.<br />
*<em>Calibration curve</em><br />
**A calibration curve was obtained, by inducing in triplicate wells of <partinfo>BBa_T9002</partinfo> with different known HSL concentrations:<br />
***10 uM<br />
***1 uM<br />
***100 nM<br />
*** 50 nM<br />
*** 10 nM<br />
*** 5 nM<br />
***2 nM<br />
***1 nM<br />
*** 0,5 nM<br />
*** 0,1 nM<br />
*** 0 M<br />
**All inductions were performed adding 2ul of "inducer solution" (3OC6-HSL, Sigma Aldrich) at a proper concentration in 200 ul of culture.<br />
*<em>HSL quantification</em><br />
**2ul of supernatants prepared as described above were used to induce 200 ul of <partinfo>BBa_T9002</partinfo> cultures in triplicate. <br />
**If the amount of 3OC6-HSL present in the supernatant was not enough concentrated to trigger the induction of <partinfo>BBa_T9002</partinfo>, a bigger amount of supernatant (X ul) was used to induce the cultures (200-X ul of cultures). In this way, the calibration curve was obtained by using the same amount of <partinfo>BBa_T9002</partinfo> culture (200-X ul).<br />
**Fluorescence and absorbance were measured after 30 min from the induction with a Tecan Infinite F200 microplate reader and, using the information provided by the calibration curve, the amount of 3OC6-HSL present in every supernatant was evaluated (each reported fluorescence value was corrected with the O.D.600 of the culture).<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
<br />
===Microplate reader experiments for <partinfo>BBa_F2620</partinfo> - Protocol #4===<br />
<br />
This protocol is identical to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|this one]], but in addition, after cultures were transferred in the 96-well microplate, 2ul of inducer solution at the proper concentration were added to each well, thus obtaining the final desired concentration.<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
=Data Analysis=<br />
<br><br />
===Preliminary remarks===<br />
*All our growth curves have been obtained subtracting for each time sample the broth O.D.600 measurement from that of the culture; broth was considered in the same conditions of the culture (e.g. induced with the same inducer concentration and supplemented with the same antibiotic of the culture).<br />
<br />
*Fluorescence signals have been obtained subtracting for each time sample the fluorescent measurement of a non-fluorescent culture from that of the target culture. The non-fluorescent culture was considered in the same conditions of the target (e.g. induced with the same inducer concentration and with the BioBrick carried by the same plasmid, not encoding a fluorescent protein). This operation allows the removal, from the target fluorescent signal, of the "self-fluorescent" component and the fluorescence signal obtained is "blanked".<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Doubling time evaluation===<br />
The natural logarithm of the growth curves (processed according to the above section) was computed and the linear phase (corresponding to the bacterial exponential growth phase) was isolated by visual inspection. Then the linear regression was performed in order to estimate the slope of the line m. Finally the doubling time was estimated as d=ln(2)/m [minutes].<br />
<br />
In the case of multiple growth curves for a strain, the mean value of the processed curves was computed for each time sample before applying the above described procedure. <br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis for self-inducible promoters (initiation-treshold determination)===<br />
<br />
The task of our analysis is the evaluation of the initiation transcription point (in terms of absorbance) for self-inducible devices. The transition O.D.600 value is named, from now on, ODstart.<br />
Data from three indipendent wells were averaged and blanked. O.D.600 signals were blanked as described in "Preliminary Remarks" section, while the fluorescence signal was blanked with the fluorescence of <partinfo>BBa_T9002</partinfo> part, assembled in the same plasmid of the considered promoter. This operation allows the removal from the fluorescence signal of the autofluorescence component and of the “leaky ” component (due to the leakage of pLux in absence of the autoinducer molecule HSL).<br />
<br />
ODstart was evaluated by computing the Scell signal for the desired self-inducible promoter and for the negative control.<br />
Two different signals, measured from independent samples of the same non-fluorescent culture in the same experiment, are considered: C1 and C2. The fluorescence signal of C1 and C2 can be thought as the addition of a “real signal” and of a noise component.<br />
<br />
[[Image:UNIPV_Pavia_noise1.png|400px|center]]<br />
<br />
since they are two time series acquired from the same culture, in the same growth condition by the same instrument.<br />
<br />
F_C1 and F_C2 have the same expected value and the same standard deviation, since they are two independent realizations of the same aleatory process: in fact, they are two time series acquired from the same cultures, in the same growth conditions by the same instrument.<br />
Noise signal was computed as:<br />
<br />
[[Image:UNIPV_Pavia_noise2.png|150px|center]]<br />
<br />
The behaviour of the signal N is shown in figure:<br />
[[Image:UNIPV_Pavia_rumoreNoise.png|600px|center|Measurement Noise]]<br />
<br />
An interesting signal is Scell of N. It can be evaluated as the time derivative of N, divided by O.D.600 of the culture. It has the behaviour shown in figure:<br />
<br />
[[Image:UNIPV_Pavia_rumoreNoiseScell.png|600px|center|Scell Noise]]<br />
It is fair to say that the noise of Scell is bigger for low O.D.600 values and its width decreases dramatically for higher O.D.600 values (e.g.: O.D.600>0,1 TECAN infinite F200).<br />
<br />
The noise model proposed for the Scell noise signal is:<br />
<br />
[[Image:UNIPV_Pavia_ModelNoiseScell.png|300px|center|Scell noise model]]<br />
<br />
Once derived the noise of the Scell signal, the evaluation of the O.D.start can be performed supposing that the signal is significantly growing when at least 5 consecutive time samples exceed a threshold defined as follows:<br />
<br />
[[Image:UNIPV_Pavia_threshold.png|300px|center|variance threshold]]<br />
<br />
The operation of subtracting the two signals F_C1 and F_C2 is analogous to the “blanking” operation performed on data, as described previously. For, this reason, under the hypothesis that the signals have the same variance (this is a consistent hypothesis, since they are measured by the same instrument in the same experimental conditions), this argument can be extended to the “blanked” data considered in the processing. <br />
In particular, it is evident that the "blanked" signal is affected by the same noise described above and thus, the noise of SCell signal is analogous to the one described before.<br />
Thus, this threshold was used to compute the O.D.start for the cultures. This heuristic algorithm was implemented in Matlab and analysis results are reported in the “results” section.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
<br />
===Data Analysis to estimate the HSL synthesys rate per cell===<br />
<br />
<br />
The autoinducer synthesys rate per cell is a very important parameter to be evaluated in quorum sensing systems. <br />
In fact the knowledge of this parameter enables the rational design of cell-communication systems, such as the self inducible devices studied in this project.<br />
<br />
A model based approach was proposed to estimate this interesting parameter. <br />
<br />
Under the hypotheses that:<br />
* no HSL is present at the beginning of the experiment - consistent hypothesis because a growth medium washing step is always performed before the experiment start in order to remove the HSL produced until that moment,<br />
* the HSL synthesis rate is not time-dependent (cells produce HSL at the same rate in each growth phase - this hypotesis simplifies the data analysis, but it should be further validated),<br />
* the half life of the autoinducer is much longer than the experiment duration (i.e. the degradation of the molecule is negligible - this hypothesis is consistent, since no HSL-degrading enzyme is present in ''E. coli''),<br />
<br />
an Ordinary Differential Equation (ODE) model which describes the HSL production in the growth media can be written:<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula1.png|250px]]<br />
|}<br />
where:<br />
* [HSL(t)] is the concentration of HSL molecule in the growth medium;<br />
* O.D.600(t) is the growth curve of the considered culture;<br />
* N is the Colony Forming Units (CFU) per O.D.600 unit (i.e. CFU in a well = O.D.600 * N)<br />
* K_HSL is the 3OC6-HSL synthesis rate per cell<br />
<br />
<br />
The analytical solution of this system is:<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula2.png|300px]]<br />
|}<br />
K_HSL can be estimated for t=t_bar, corresponding to the trasncription initiation time, as reported below.<br />
<br />
{| align='center'<br />
|[[Image:PV_Immagine_formula3.png|250px]]<br />
|}<br />
where:<br />
* N was estimated by counting TOP10 colonies obtained by plating serial dilutions of cultures with known O.D.600.<br />
* [HSL(t_bar)] was estimated as described in the section [[Data Analysis - minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>]]<br />
* the O.D.600 time series was measured in each experiment, so its time integral can be computed by trapezoidal numerical integration.<br />
<br />
N was estimated and values are reported below:<br />
<br />
{| border='1' align='center'<br />
| || &nbsp;&nbsp;&nbsp;'''LB'''&nbsp;&nbsp;&nbsp; || &nbsp;&nbsp;&nbsp;'''M9'''&nbsp;&nbsp;&nbsp;<br />
|-<br />
| &nbsp; &nbsp; &nbsp; '''N''' &nbsp; &nbsp; &nbsp;|| &nbsp;&nbsp;2,818 * 10^9 &nbsp;&nbsp;||&nbsp;&nbsp; 2,123 * 10^9 &nbsp;&nbsp;<br />
|}<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data Analysis - minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>===<br />
<br />
Cultures were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for BBa_F2620 - Protocol #4|this protocol]].<br />
<br />
Data from three indipendent wells were averaged and blanked as described in "Preliminary Remarks" section.<br />
<br />
Minimum induction required to activate ''lux pR'' was evaluated by a visual inspection of Scell signal<br />
<br />
<br />
[[Image:pv_MinimumInduction.png|330px|thumb|center|Minimum induction required to activate ''lux pR'' for <partinfo>BBa_F2620</partinfo>]]<br />
<br />
<br />
<div align='center'><br />
'''HSL(t_bar)''' : 0,04 nM<br />
</div><br />
HSL(t_bar) was evaluated only in M9 medium because it is a minimum autofluorescence growth medium and it provides the most accurate result in estimating a low fluorescence.<br />
<br />
<br />
<br />
<br />
<br><br />
----<br />
<br><br />
<br />
===Data analysis for RPU evaluation===<br />
<br />
The RPUs are standard units proposed by Kelly J. et al., 2008, in which the relative transcriptional strength of a promoter can be measured using a reference standard.<br />
<br />
RPUs have been computed as:<br />
[[Image:UNIPV_Pavia_RPU_formula.png|400px|center]]<br />
<br />
in which:<br />
* phi is the promoter of interest and J23101 is the reference standard promoter (taken from the Anderson Promoter Collection);<br />
* F is the blanked fluorescence of the culture, computed by subtracting for each time sample the fluorescence value of a negative control (a non-fluorescent culture). In our experiments, the TOP10 cells bearing BBa_B0032 or BBa_B0033 were usually used because they are RBSs and do not have expression systems for reporter genes;<br />
* ASB is the blanked absorbance (O.D.600) of the culture, computed as described in "Preliminary remarks" section. <br />
RPU measurement has the following advantages (under suitable conditions)<br />
* it is proportional to PoPS (Polymerase Per Second), a very important parameter that expresses the transcription rate of a promoter;<br />
* it uses a reference standard and so measurements can be compared between different laboratories. <br />
The hypotheses on which RPU theory is based can be found in Kelly J. et al., 2008, as well as all the mathematical steps. From our point of view, the main hypotheses that have to be satisfied are the following:<br />
* the reporter protein must have a half life higher than the experiment duration (we use GFPmut3 - <partinfo>BBa_E0040</partinfo> -, which has an estimated half life of at least 24 hours, or an engineered RFP - <partinfo>BBa_E1010</partinfo>, for which the half life has not been measured, but is qualitatively comparable with the GFP's);<br />
* strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in J23101 reference standard.<br />
* steady state must be valid, so (dF/dt)/ASB (proportional to the GFP synthesis rate per cell) must be constant.<br />
<br />
In order to compute the RPUs, the Scell signals ((dGFP/dt)/ASB)) of the promoter of interest and of the reference J23101 were averaged in the time interval corresponding to the exponential growth phase. The boundaries of exponential phase were identified with a visual inspection of the linear phase of the logarithmic growth curve.<br />
<br />
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<br />
</td></tr></table><br />
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[[Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry |Existing Parts from the Registry]]<hr><br />
</td></tr></table> --></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAutoTeam:UNIPV-Pavia/Project/PromotoriAuto2010-10-27T14:14:27Z<p>Susanna: /* Results */</p>
<hr />
<div>__NOTOC__<br />
{{UNIPV-Pavia/header}} <br />
<br />
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<table width="100%" border="0" align="center"><br />
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<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
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<font size="5" face="Cataneo BT" align="center" valign="top">'''''ProteInProgress: a cellular assembly line for protein manufacturing'''''</font><br />
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/motivation"><br />
<img src="https://static.igem.org/mediawiki/2010/b/be/UNIPV_Pavia_motivation2_BN.jpg" width="75px" height="75px" alt="Motivation" title="Motivation"/><br />
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/results"><br />
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/references"><br />
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<p align="center" valign="top"><br><br />
<font size="5" align="center" valign="top"><b>Implementation and Results: Self-Inducible promoters</b></font><br />
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<br><br />
[[Team:UNIPV-Pavia/Project/PromotoriAuto|Self-inducible promoters]]<br />
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntColi"><br />
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<br><br />
[[Team:UNIPV-Pavia/Project/IntColi|Integrative standard vector for E. coli]]<br />
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntYeast"><br />
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<br><br />
[[Team:UNIPV-Pavia/Project/IntYeast|Integrative standard vector for yeast]]<br />
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/FasinaInteina"><br />
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[[Team:UNIPV-Pavia/Project/FasinaInteina|Purification of proteins]]<br />
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<br />
=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein production</b>===<br />
<br />
'''Experimental implementation:''' <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:<br />
<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>Strength (a.u.)<br>reported in the Registry</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 2547<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||1791<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||623<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||1185<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||844<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||256<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||396<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||1429<br />
|}<br />
<br />
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]], using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:<br />
*high copy number plasmids and LB;<br />
*high copy number plasmids and M9;<br />
*low copy number plasmids and M9.<br />
It was not possible to evaluate promoters activities in low copy number plasmids in LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2]] and data were analyzed as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]]; <br />
<br />
'''Results''': results are shown here.<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI the <partinfo>BBa_J61002</partinfo>-BBa_J231xx EcoRI-PstI fragment in <partinfo>pSB4C5</partinfo>, in order to transfer the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
<br />
Doubling times were evaluated for the described cultures:<br />
{| align='center' border='1'<br />
|rowspan='2'|<b>Promoter</b><br />
|colspan='3'| <b>doubling time [minutes]</b><br />
|-<br />
| LB in HC plasmid || M9 in HC plasmid || M9 in LC plasmid<br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || || ||<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>|| || ||<br />
|}<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced LuxI protein amounts that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformans could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluoresent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced</b>===<br />
<br />
'''Experimental implementation''' The new parts were, thus, characterized, measuring the HSL concentration released in the medium after a 6 hour growth of the cultures. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 sensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts contained in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in high copy plasmid]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced after 6 hours growth by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> is reported in Fig.9 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in low copy plasmid]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) in high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized here:<br />
<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan="2" align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center' width='80%'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained <br>in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|300px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|300px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|300px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|300px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]]<br />
*K_HSL is the HSL synthesys rate per cell, it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis to estimate the HSL synthesys rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double. It was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
<br>[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> ± <br> *<br />
| 8.94 10^-17 <br> ± <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> ± <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> ± <br> *<br />
| 7.53 10^-18 <br> ± <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> ± <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br>[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
<br />
'''Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br><br />
<table><tr><td>[[Image:pv_BBa_K300030.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
|Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br><br />
<table><tr><td>[[Image:pv_BBa_K300028.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300029</partinfo> (wiki name: I16) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br><br />
<table><tr><td>[[Image:pv_BBa_K300029.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.31 <br> ± <br> *<br />
| 1.17 10^-16 <br> ± <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> ± <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br><br />
<table><tr><td>[[Image:pv_BBa_K300025.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br><br />
<table><tr><td>[[Image:pv_BBa_K300026.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br><br />
<table><tr><td>[[Image:pv_BBa_K300027.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 700px; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two self-inducible devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were realized and characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell and an algorithm was proposed, in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In figure, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported, both for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid). <br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the Signal Molucule production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by data. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate results stronger at the increase of promoter's strength. <br />
<br />
For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably due to the fact that too high synthesis rate of HSL are injurious for the cell. <br />
A range of RPU values for promoters that control the Sender device was identified. When the PoPs-in signal belongs to this range, the expected behaviour is confirmed by experimental data. <br />
The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices, able to start the production of a target protein at different O.D._start values, in any cellular growth phase.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAutoTeam:UNIPV-Pavia/Project/PromotoriAuto2010-10-27T14:09:43Z<p>Susanna: </p>
<hr />
<div>__NOTOC__<br />
{{UNIPV-Pavia/header}} <br />
<br />
<br />
<table width="100%" border="0" align="center"><br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td align="justify" valign="top" style="padding:20px"><br />
<p align="center" valign="top"><br><br />
<font size="5" face="Cataneo BT" align="center" valign="top">'''''ProteInProgress: a cellular assembly line for protein manufacturing'''''</font><br />
</p><br />
<hr><br />
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<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/motivation"><br />
<img src="https://static.igem.org/mediawiki/2010/b/be/UNIPV_Pavia_motivation2_BN.jpg" width="75px" height="75px" alt="Motivation" title="Motivation"/><br />
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/results"><br />
<img src="https://static.igem.org/mediawiki/2010/7/77/UNIPV_Pavia_result.jpg" width="75px" height="75px" alt="Implementation & Results" title="Implementation & Results"/><br />
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/references"><br />
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</table><br />
<br />
<html><a name="indice"/></a></html><br />
<br />
<p align="center" valign="top"><br><br />
<font size="5" align="center" valign="top"><b>Implementation and Results: Self-Inducible promoters</b></font><br />
</p><br />
<br><br />
<hr><br />
<br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
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<br><br />
[[Team:UNIPV-Pavia/Project/PromotoriAuto|Self-inducible promoters]]<br />
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntColi"><br />
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<br><br />
[[Team:UNIPV-Pavia/Project/IntColi|Integrative standard vector for E. coli]]<br />
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntYeast"><br />
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[[Team:UNIPV-Pavia/Project/IntYeast|Integrative standard vector for yeast]]<br />
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/FasinaInteina"><br />
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[[Team:UNIPV-Pavia/Project/FasinaInteina|Purification of proteins]]<br />
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=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein production</b>===<br />
<br />
'''Experimental implementation:''' <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:<br />
<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>Strength (a.u.)<br>reported in the Registry</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 2547<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||1791<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||623<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||1185<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||844<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||256<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||396<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||1429<br />
|}<br />
<br />
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]], using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:<br />
*high copy number plasmids and LB;<br />
*high copy number plasmids and M9;<br />
*low copy number plasmids and M9.<br />
It was not possible to evaluate promoters activities in low copy number plasmids in LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2]] and data were analyzed as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]]; <br />
<br />
'''Results''': results are shown here.<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI the <partinfo>BBa_J61002</partinfo>-BBa_J231xx EcoRI-PstI fragment in <partinfo>pSB4C5</partinfo>, in order to transfer the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
<br />
Doubling times were evaluated for the described cultures:<br />
{| align='center' border='1'<br />
|rowspan='2'|<b>Promoter</b><br />
|colspan='3'| <b>doubling time [minutes]</b><br />
|-<br />
| LB in HC plasmid || M9 in HC plasmid || M9 in LC plasmid<br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || || ||<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>|| || ||<br />
|}<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced LuxI protein amounts that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformans could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluoresent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced</b>===<br />
<br />
'''Experimental implementation''' The new parts were, thus, characterized, measuring the HSL concentration released in the medium after a 6 hour growth of the cultures. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 sensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts contained in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in high copy plasmid]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced after 6 hours growth by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> is reported in Fig.9 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in low copy plasmid]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) in high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized here:<br />
<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan="2" align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|400px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|400px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|400px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|400px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]]<br />
*K_HSL is the HSL synthesys rate per cell, it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis to estimate the HSL synthesys rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double. It was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
<br>[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> ± <br> *<br />
| 8.94 10^-17 <br> ± <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> ± <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> ± <br> *<br />
| 7.53 10^-18 <br> ± <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> ± <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br>[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br>[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
<br />
'''Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1' width='80%'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br><br />
<table><tr><td>[[Image:pv_BBa_K300030.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
|Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br><br />
<table><tr><td>[[Image:pv_BBa_K300028.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300029</partinfo> (wiki name: I16) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br><br />
<table><tr><td>[[Image:pv_BBa_K300029.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| 0.31 <br> ± <br> *<br />
| 1.17 10^-16 <br> ± <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> ± <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br><br />
<table><tr><td>[[Image:pv_BBa_K300025.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br><br />
<table><tr><td>[[Image:pv_BBa_K300026.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
<br><br />
<table><tr><td>[[Image:pv_BBa_K300027.png|170px]]<br>LC</td><br />
<td>[[Image:pv_BBa_F2620.png|170px]]<br>HC</td></tr></table><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 700px; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two self-inducible devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were realized and characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell and an algorithm was proposed, in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In figure, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported, both for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid). <br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the Signal Molucule production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by data. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate results stronger at the increase of promoter's strength. <br />
<br />
For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably due to the fact that too high synthesis rate of HSL are injurious for the cell. <br />
A range of RPU values for promoters that control the Sender device was identified. When the PoPs-in signal belongs to this range, the expected behaviour is confirmed by experimental data. <br />
The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices, able to start the production of a target protein at different O.D._start values, in any cellular growth phase.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAutoTeam:UNIPV-Pavia/Project/PromotoriAuto2010-10-27T12:46:25Z<p>Susanna: /* Regulation of signal protein production */</p>
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<div>__NOTOC__<br />
{{UNIPV-Pavia/header}} <br />
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<font size="5" face="Cataneo BT" align="center" valign="top">'''''ProteInProgress: a cellular assembly line for protein manufacturing'''''</font><br />
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<font size="5" align="center" valign="top"><b>Implementation and Results: Self-Inducible promoters</b></font><br />
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[[Team:UNIPV-Pavia/Project/PromotoriAuto|Self-inducible promoters]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntColi"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/UNIPV_Pavia_Icona_intColi.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntColi|Integrative standard vector for E. coli]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntYeast"><br />
<img src="https://static.igem.org/mediawiki/2010/0/03/UNIPV_Pavia_Icona_IntYeast.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntYeast|Integrative standard vector for yeast]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/FasinaInteina"><br />
<img src="https://static.igem.org/mediawiki/2010/7/74/UNIPV_Pavia_Icona_Bioplastica.GIF" width="75px" height="75px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/FasinaInteina|Purification of proteins]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/><br />
</html><br />
<br />
=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein production</b>===<br />
<br />
'''Experimental implementation:''' <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:<br />
<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>Strength (a.u.)<br>reported in the Registry</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 2547<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||1791<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||623<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||1185<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||844<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||256<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||396<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||1429<br />
|}<br />
<br />
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]], using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:<br />
*high copy number plasmids and LB;<br />
*high copy number plasmids and M9;<br />
*low copy number plasmids and M9.<br />
It was not possible to evaluate promoters activities in low copy number plasmids and LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2]] and data were analyzed as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]]; <br />
<br />
'''Results''': results are shown here.<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI the <partinfo>BBa_J61002</partinfo>-BBa_J231xx EcoRI-PstI fragment in <partinfo>pSB4C5</partinfo>, in order to transfer the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
<br />
Doubling time were evaluated for the described cultures:<br />
{| align='center' border='1'<br />
|rowspan='2'|<b>Promoter</b><br />
|colspan='3'| <b>doubling time [minutes]</b><br />
|-<br />
| LB in HC plasmid || M9 in HC plasmid || M9 in LC plasmid<br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || || ||<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>|| || ||<br />
|}<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced LuxI protein amounts that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformans could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluoresent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced</b>===<br />
<br />
'''Experimental implementation''' The new parts were, thus, characterized, measuring the HSL concentration released in the medium after a 6 hour growth of the cultures. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 sensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts contained in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in high copy plasmid]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced after 6 hours growth by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> is reported in Fig.9 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in low copy plasmid]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) beared on high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized here:<br />
<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan="2" align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|400px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|400px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|400px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|400px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]]<br />
*K_HSL is the HSL synthesys rate per cell, it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis to estimate the HSL synthesys rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double. It was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
|[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> ± <br> *<br />
| 8.94 10^-17 <br> ± <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> ± <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> ± <br> *<br />
| 7.53 10^-18 <br> ± <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> ± <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
'''Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center' colspan='2'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300030.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300028.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300029</partinfo><br> (wiki name: I16)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300029.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| 0.31 <br> ± <br> *<br />
| 1.17 10^-16 <br> ± <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> ± <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300025.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300026.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300027.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 700px; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two self-inducible devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were realized and characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell and an algorithm was proposed, in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In figure, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported, both for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid). <br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the Signal Molucule production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by data. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate results stronger at the increase of promoter's strength. <br />
<br />
For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably due to the fact that too high synthesis rate of HSL are injurious for the cell. <br />
A range of RPU values for promoters that control the Sender device was identified. When the PoPs-in signal belongs to this range, the expected behaviour is confirmed by experimental data. <br />
The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices, able to start the production of a target protein at different O.D._start values, in any cellular growth phase.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAutoTeam:UNIPV-Pavia/Project/PromotoriAuto2010-10-27T12:41:17Z<p>Susanna: /* Regulation of signal protein production */</p>
<hr />
<div>__NOTOC__<br />
{{UNIPV-Pavia/header}} <br />
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<font size="5" face="Cataneo BT" align="center" valign="top">'''''ProteInProgress: a cellular assembly line for protein manufacturing'''''</font><br />
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<font size="5" align="center" valign="top"><b>Implementation and Results: Self-Inducible promoters</b></font><br />
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<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAuto"><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/PromotoriAuto|Self-inducible promoters]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntColi"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/UNIPV_Pavia_Icona_intColi.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntColi|Integrative standard vector for E. coli]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntYeast"><br />
<img src="https://static.igem.org/mediawiki/2010/0/03/UNIPV_Pavia_Icona_IntYeast.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntYeast|Integrative standard vector for yeast]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/FasinaInteina"><br />
<img src="https://static.igem.org/mediawiki/2010/7/74/UNIPV_Pavia_Icona_Bioplastica.GIF" width="75px" height="75px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/FasinaInteina|Purification of proteins]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/><br />
</html><br />
<br />
=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein production</b>===<br />
<br />
'''Experimental implementation:''' <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:<br />
<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>Strength (a.u.)<br>reported in the Registry</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 2547<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||1791<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||623<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||1185<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||844<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||256<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||396<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||1429<br />
|}<br />
<br />
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]], using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:<br />
*high copy number plasmids and LB;<br />
*high copy number plasmids and M9;<br />
*low copy number plasmids and M9.<br />
It was not possible to evaluate promoters activities in low copy number plasmids and LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2]] and data were analyzed as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]]; <br />
<br />
'''Results''': results are shown here.<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI the <partinfo>BBa_J61002</partinfo>-BBa_J231xx EcoRI-PstI fragment in <partinfo>pSB4C5</partinfo>, in order to transfer the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
<br />
Doubling time were evaluated for the described cultures:<br />
{| align='center' border='1'<br />
|colspan='2'|<b>Promoter</b><br />
|rowspan='3'| <b>doubling time [minutes]</b><br />
|-<br />
| LB in HC plasmid || M9 in HC plasmid || M9 in LC plasmid<br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || || ||<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>|| || ||<br />
|}<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced LuxI protein amounts that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformans could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluoresent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced</b>===<br />
<br />
'''Experimental implementation''' The new parts were, thus, characterized, measuring the HSL concentration released in the medium after a 6 hour growth of the cultures. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 sensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts contained in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in high copy plasmid]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced after 6 hours growth by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> is reported in Fig.9 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in low copy plasmid]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) beared on high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized here:<br />
<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan="2" align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|400px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|400px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|400px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|400px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]]<br />
*K_HSL is the HSL synthesys rate per cell, it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis to estimate the HSL synthesys rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double. It was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
|[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> ± <br> *<br />
| 8.94 10^-17 <br> ± <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> ± <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> ± <br> *<br />
| 7.53 10^-18 <br> ± <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> ± <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
'''Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center' colspan='2'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300030.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300028.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300029</partinfo><br> (wiki name: I16)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300029.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| 0.31 <br> ± <br> *<br />
| 1.17 10^-16 <br> ± <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> ± <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300025.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300026.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300027.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 700px; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two self-inducible devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were realized and characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell and an algorithm was proposed, in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In figure, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported, both for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid). <br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the Signal Molucule production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by data. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate results stronger at the increase of promoter's strength. <br />
<br />
For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably due to the fact that too high synthesis rate of HSL are injurious for the cell. <br />
A range of RPU values for promoters that control the Sender device was identified. When the PoPs-in signal belongs to this range, the expected behaviour is confirmed by experimental data. <br />
The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices, able to start the production of a target protein at different O.D._start values, in any cellular growth phase.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAutoTeam:UNIPV-Pavia/Project/PromotoriAuto2010-10-27T12:37:44Z<p>Susanna: /* Regulation of signal protein production */</p>
<hr />
<div>__NOTOC__<br />
{{UNIPV-Pavia/header}} <br />
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<font size="5" face="Cataneo BT" align="center" valign="top">'''''ProteInProgress: a cellular assembly line for protein manufacturing'''''</font><br />
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</table><br />
<br />
<html><a name="indice"/></a></html><br />
<br />
<p align="center" valign="top"><br><br />
<font size="5" align="center" valign="top"><b>Implementation and Results: Self-Inducible promoters</b></font><br />
</p><br />
<br><br />
<hr><br />
<br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAuto"><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/PromotoriAuto|Self-inducible promoters]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntColi"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/UNIPV_Pavia_Icona_intColi.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntColi|Integrative standard vector for E. coli]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntYeast"><br />
<img src="https://static.igem.org/mediawiki/2010/0/03/UNIPV_Pavia_Icona_IntYeast.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntYeast|Integrative standard vector for yeast]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/FasinaInteina"><br />
<img src="https://static.igem.org/mediawiki/2010/7/74/UNIPV_Pavia_Icona_Bioplastica.GIF" width="75px" height="75px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/FasinaInteina|Purification of proteins]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/><br />
</html><br />
<br />
=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein production</b>===<br />
<br />
'''Experimental implementation:''' <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:<br />
<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>Strength (a.u.)<br>reported in the Registry</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 2547<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||1791<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||623<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||1185<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||844<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||256<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||396<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||1429<br />
|}<br />
<br />
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]], using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:<br />
*high copy number plasmids and LB;<br />
*high copy number plasmids and M9;<br />
*low copy number plasmids and M9.<br />
It was not possible to evaluate promoters activities in low copy number plasmids and LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2]] and data were analyzed as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]]; <br />
<br />
'''Results''': results are shown here.<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI the <partinfo>BBa_J61002</partinfo>-BBa_J231xx EcoRI-PstI fragment in <partinfo>pSB4C5</partinfo>, in order to transfer the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
<br />
Doubling time were evaluated for the described cultures:<br />
{| align='center' border='1'<br />
|colspan='2'|<b>Promoter</b> |rowspan='3'| <b>doubling time [minutes]</b><br />
|-<br />
| LB in HC plasmid || M9 in HC plasmid || M9 in LC plasmid<br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || || ||<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>|| || ||<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>|| || ||<br />
|}<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced LuxI protein amounts that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformans could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluoresent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced</b>===<br />
<br />
'''Experimental implementation''' The new parts were, thus, characterized, measuring the HSL concentration released in the medium after a 6 hour growth of the cultures. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 sensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts contained in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in high copy plasmid]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced after 6 hours growth by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> is reported in Fig.9 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in low copy plasmid]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) beared on high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized here:<br />
<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan="2" align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|400px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|400px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|400px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|400px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]]<br />
*K_HSL is the HSL synthesys rate per cell, it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis to estimate the HSL synthesys rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double. It was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
|[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> ± <br> *<br />
| 8.94 10^-17 <br> ± <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> ± <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> ± <br> *<br />
| 7.53 10^-18 <br> ± <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> ± <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
'''Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center' colspan='2'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300030.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300028.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300029</partinfo><br> (wiki name: I16)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300029.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| 0.31 <br> ± <br> *<br />
| 1.17 10^-16 <br> ± <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> ± <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300025.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300026.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300027.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 700px; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two self-inducible devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were realized and characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell and an algorithm was proposed, in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In figure, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported, both for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid). <br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the Signal Molucule production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by data. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate results stronger at the increase of promoter's strength. <br />
<br />
For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably due to the fact that too high synthesis rate of HSL are injurious for the cell. <br />
A range of RPU values for promoters that control the Sender device was identified. When the PoPs-in signal belongs to this range, the expected behaviour is confirmed by experimental data. <br />
The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices, able to start the production of a target protein at different O.D._start values, in any cellular growth phase.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAutoTeam:UNIPV-Pavia/Project/PromotoriAuto2010-10-27T12:30:35Z<p>Susanna: </p>
<hr />
<div>__NOTOC__<br />
{{UNIPV-Pavia/header}} <br />
<br />
<br />
<table width="100%" border="0" align="center"><br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td align="justify" valign="top" style="padding:20px"><br />
<p align="center" valign="top"><br><br />
<font size="5" face="Cataneo BT" align="center" valign="top">'''''ProteInProgress: a cellular assembly line for protein manufacturing'''''</font><br />
</p><br />
<hr><br />
<br><br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/motivation"><br />
<img src="https://static.igem.org/mediawiki/2010/b/be/UNIPV_Pavia_motivation2_BN.jpg" width="75px" height="75px" alt="Motivation" title="Motivation"/><br />
</a><br />
</html></td><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/solution"><br />
<img src="https://static.igem.org/mediawiki/2010/c/c8/UNIPV_Pavia_solution2_BN.jpg" width="75px" height="75px" alt="Solutions" title="Solutions"/></a><br />
</html></td><br />
<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/results"><br />
<img src="https://static.igem.org/mediawiki/2010/7/77/UNIPV_Pavia_result.jpg" width="75px" height="75px" alt="Implementation & Results" title="Implementation & Results"/><br />
</a><br />
</html></td><br />
<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/references"><br />
<img src="https://static.igem.org/mediawiki/2010/6/61/UNIPV_Paviareferences_BN.jpg" width="75px" height="75px" alt="References" title="References"/></a><br />
</html></td><br />
</tr><br />
</table><br />
<br />
<html><a name="indice"/></a></html><br />
<br />
<p align="center" valign="top"><br><br />
<font size="5" align="center" valign="top"><b>Implementation and Results: Self-Inducible promoters</b></font><br />
</p><br />
<br><br />
<hr><br />
<br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAuto"><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/PromotoriAuto|Self-inducible promoters]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntColi"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/UNIPV_Pavia_Icona_intColi.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntColi|Integrative standard vector for E. coli]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntYeast"><br />
<img src="https://static.igem.org/mediawiki/2010/0/03/UNIPV_Pavia_Icona_IntYeast.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntYeast|Integrative standard vector for yeast]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/FasinaInteina"><br />
<img src="https://static.igem.org/mediawiki/2010/7/74/UNIPV_Pavia_Icona_Bioplastica.GIF" width="75px" height="75px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/FasinaInteina|Purification of proteins]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/><br />
</html><br />
<br />
=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein production</b>===<br />
<br />
'''Experimental implementation:''' <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:<br />
<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>Strength (a.u.)<br>reported in the Registry</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 2547<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||1791<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||623<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||1185<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||844<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||256<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||396<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||1429<br />
|}<br />
<br />
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]], using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:<br />
*high copy number plasmids and LB;<br />
*high copy number plasmids and M9;<br />
*low copy number plasmids and M9.<br />
It was not possible to evaluate promoters activities in low copy number plasmids and LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2]] and data were analyzed as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]]; <br />
<br />
'''Results''': results are shown here.<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI the <partinfo>BBa_J61002</partinfo>-BBa_J231xx EcoRI-PstI fragment in <partinfo>pSB4C5</partinfo>, in order to transfer the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
<br />
Doubling time were evaluated for the described cultures:<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>doubling time [minutes]</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || <br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||<br />
|}<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced LuxI protein amounts that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformans could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluoresent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced</b>===<br />
<br />
'''Experimental implementation''' The new parts were, thus, characterized, measuring the HSL concentration released in the medium after a 6 hour growth of the cultures. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 sensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts contained in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in high copy plasmid]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced after 6 hours growth by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> is reported in Fig.9 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in low copy plasmid]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) beared on high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized here:<br />
<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan="2" align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|400px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|400px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|400px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|400px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]]<br />
*K_HSL is the HSL synthesys rate per cell, it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis to estimate the HSL synthesys rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double. It was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
|[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> ± <br> *<br />
| 8.94 10^-17 <br> ± <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> ± <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> ± <br> *<br />
| 7.53 10^-18 <br> ± <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> ± <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
'''Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center' colspan='2'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300030.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300028.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300029</partinfo><br> (wiki name: I16)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300029.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| 0.31 <br> ± <br> *<br />
| 1.17 10^-16 <br> ± <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> ± <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300025.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300026.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300027.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 700px; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two self-inducible devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were realized and characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell and an algorithm was proposed, in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In figure, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported, both for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid). <br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the Signal Molucule production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by data. This is consistent with the expected behaviour of these parts, since the HSL synthesis rate results stronger at the increase of promoter's strength. <br />
<br />
For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably due to the fact that too high synthesis rate of HSL are injurious for the cell. <br />
A range of RPU values for promoters that control the Sender device was identified. When the PoPs-in signal belongs to this range, the expected behaviour is confirmed by experimental data. <br />
The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices, able to start the production of a target protein at different O.D._start values, in any cellular growth phase.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div></div>Susannahttp://2010.igem.org/File:Pv_RPUvsOD_HCHC_M9.pngFile:Pv RPUvsOD HCHC M9.png2010-10-27T11:00:08Z<p>Susanna: uploaded a new version of "Image:Pv RPUvsOD HCHC M9.png"</p>
<hr />
<div></div>Susannahttp://2010.igem.org/File:Pv_RPUvsOD_HCLC_M9.pngFile:Pv RPUvsOD HCLC M9.png2010-10-27T11:00:05Z<p>Susanna: uploaded a new version of "Image:Pv RPUvsOD HCLC M9.png"</p>
<hr />
<div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAutoTeam:UNIPV-Pavia/Project/PromotoriAuto2010-10-27T10:54:54Z<p>Susanna: /* Results */</p>
<hr />
<div>__NOTOC__<br />
{{UNIPV-Pavia/header}} <br />
<br />
<br />
<table width="100%" border="0" align="center"><br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td align="justify" valign="top" style="padding:20px"><br />
<p align="center" valign="top"><br><br />
<font size="5" face="Cataneo BT" align="center" valign="top">'''''ProteInProgress: a cellular assembly line for protein manufacturing'''''</font><br />
</p><br />
<hr><br />
<br><br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/motivation"><br />
<img src="https://static.igem.org/mediawiki/2010/b/be/UNIPV_Pavia_motivation2_BN.jpg" width="75px" height="75px" alt="Motivation" title="Motivation"/><br />
</a><br />
</html></td><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/solution"><br />
<img src="https://static.igem.org/mediawiki/2010/c/c8/UNIPV_Pavia_solution2_BN.jpg" width="75px" height="75px" alt="Solutions" title="Solutions"/></a><br />
</html></td><br />
<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/results"><br />
<img src="https://static.igem.org/mediawiki/2010/7/77/UNIPV_Pavia_result.jpg" width="75px" height="75px" alt="Implementation & Results" title="Implementation & Results"/><br />
</a><br />
</html></td><br />
<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/references"><br />
<img src="https://static.igem.org/mediawiki/2010/6/61/UNIPV_Paviareferences_BN.jpg" width="75px" height="75px" alt="References" title="References"/></a><br />
</html></td><br />
</tr><br />
</table><br />
<br />
<html><a name="indice"/></a></html><br />
<br />
<p align="center" valign="top"><br><br />
<font size="5" align="center" valign="top"><b>Implementation and Results: Self-Inducible promoters</b></font><br />
</p><br />
<br><br />
<hr><br />
<br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAuto"><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/PromotoriAuto|Self-inducible promoters]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntColi"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/UNIPV_Pavia_Icona_intColi.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntColi|Integrative standard vector for E. coli]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntYeast"><br />
<img src="https://static.igem.org/mediawiki/2010/0/03/UNIPV_Pavia_Icona_IntYeast.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntYeast|Integrative standard vector for yeast]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/FasinaInteina"><br />
<img src="https://static.igem.org/mediawiki/2010/7/74/UNIPV_Pavia_Icona_Bioplastica.GIF" width="75px" height="75px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/FasinaInteina|Purification of proteins]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/><br />
</html><br />
<br />
=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein production</b>===<br />
<br />
'''Experimental implementation:''' <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:<br />
<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>Strength (a.u.)<br>reported in the Registry</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 2547<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||1791<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||623<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||1185<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||844<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||256<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||396<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||1429<br />
|}<br />
<br />
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]], using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:<br />
*high copy number plasmids and LB;<br />
*high copy number plasmids and M9;<br />
*low copy number plasmids and M9.<br />
It was not possible to evaluate promoters activities in low copy number plasmids and LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2]] and data were analyzed as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]]; <br />
<br />
'''Results''': results are shown here.<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI the <partinfo>BBa_J61002</partinfo>-BBa_J231xx EcoRI-PstI fragment in <partinfo>pSB4C5</partinfo>, in order to transfer the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
<br />
Doubling time were evaluated for the described cultures:<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>doubling time [minutes]</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || <br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||<br />
|}<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced LuxI protein amounts that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformans could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluoresent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced</b>===<br />
<br />
'''Experimental implementation''' The new parts were, thus, characterized, measuring the HSL concentration released in the medium after a 6 hour growth of the cultures. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 sensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts contained in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in high copy plasmid]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced after 6 hours growth by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> is reported in Fig.9 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in low copy plasmid]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) beared on high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized here:<br />
<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan="2" align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|400px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|400px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|400px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|400px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]]<br />
*K_HSL is the HSL synthesys rate per cell, it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis to estimate the HSL synthesys rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double. It was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
|[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> ± <br> *<br />
| 8.94 10^-17 <br> ± <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> ± <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> ± <br> *<br />
| 7.53 10^-18 <br> ± <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> ± <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
'''Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center' colspan='2'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300030.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300028.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300029</partinfo><br> (wiki name: I16)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300029.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| 0.31 <br> ± <br> *<br />
| 1.17 10^-16 <br> ± <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> ± <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300025.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300026.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300027.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 700px; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two self-inducible devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were realized and characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell and an algorithm was proposed, in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In figure, the O.D.start and the HSL synthesis rate as a function of the strength of the promoter (RPU) controlling the signal molecule production are reported, both for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid). <br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the signal molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|-<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start and K_HSL as a function of RPUs of the promoters controlling the Signal Molucule production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by data. This is consistent with the expected behavior of these parts, since the HSL synthesis rate results stronger at the increase of promoter's strength.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div></div>Susannahttp://2010.igem.org/File:Pv_RPUvsOD_HCLC_M9.pngFile:Pv RPUvsOD HCLC M9.png2010-10-27T10:51:26Z<p>Susanna: uploaded a new version of "Image:Pv RPUvsOD HCLC M9.png"</p>
<hr />
<div></div>Susannahttp://2010.igem.org/File:Pv_RPUvsOD_HCHC_M9.pngFile:Pv RPUvsOD HCHC M9.png2010-10-27T10:51:26Z<p>Susanna: uploaded a new version of "Image:Pv RPUvsOD HCHC M9.png"</p>
<hr />
<div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAutoTeam:UNIPV-Pavia/Project/PromotoriAuto2010-10-27T10:17:34Z<p>Susanna: /* Results */</p>
<hr />
<div>__NOTOC__<br />
{{UNIPV-Pavia/header}} <br />
<br />
<br />
<table width="100%" border="0" align="center"><br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td align="justify" valign="top" style="padding:20px"><br />
<p align="center" valign="top"><br><br />
<font size="5" face="Cataneo BT" align="center" valign="top">'''''ProteInProgress: a cellular assembly line for protein manufacturing'''''</font><br />
</p><br />
<hr><br />
<br><br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/motivation"><br />
<img src="https://static.igem.org/mediawiki/2010/b/be/UNIPV_Pavia_motivation2_BN.jpg" width="75px" height="75px" alt="Motivation" title="Motivation"/><br />
</a><br />
</html></td><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/solution"><br />
<img src="https://static.igem.org/mediawiki/2010/c/c8/UNIPV_Pavia_solution2_BN.jpg" width="75px" height="75px" alt="Solutions" title="Solutions"/></a><br />
</html></td><br />
<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/results"><br />
<img src="https://static.igem.org/mediawiki/2010/7/77/UNIPV_Pavia_result.jpg" width="75px" height="75px" alt="Implementation & Results" title="Implementation & Results"/><br />
</a><br />
</html></td><br />
<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/references"><br />
<img src="https://static.igem.org/mediawiki/2010/6/61/UNIPV_Paviareferences_BN.jpg" width="75px" height="75px" alt="References" title="References"/></a><br />
</html></td><br />
</tr><br />
</table><br />
<br />
<html><a name="indice"/></a></html><br />
<br />
<p align="center" valign="top"><br><br />
<font size="5" align="center" valign="top"><b>Implementation and Results: Self-Inducible promoters</b></font><br />
</p><br />
<br><br />
<hr><br />
<br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAuto"><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/PromotoriAuto|Self-inducible promoters]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntColi"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/UNIPV_Pavia_Icona_intColi.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntColi|Integrative standard vector for E. coli]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntYeast"><br />
<img src="https://static.igem.org/mediawiki/2010/0/03/UNIPV_Pavia_Icona_IntYeast.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntYeast|Integrative standard vector for yeast]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/FasinaInteina"><br />
<img src="https://static.igem.org/mediawiki/2010/7/74/UNIPV_Pavia_Icona_Bioplastica.GIF" width="75px" height="75px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/FasinaInteina|Purification of proteins]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/><br />
</html><br />
<br />
=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein production</b>===<br />
<br />
'''Experimental implementation:''' <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:<br />
<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>Strength (a.u.)<br>reported in the Registry</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 2547<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||1791<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||623<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||1185<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||844<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||256<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||396<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||1429<br />
|}<br />
<br />
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]], using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:<br />
*high copy number plasmids and LB;<br />
*high copy number plasmids and M9;<br />
*low copy number plasmids and M9.<br />
It was not possible to evaluate promoters activities in low copy number plasmids and LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2]] and data were analyzed as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]]; <br />
<br />
'''Results''': results are shown here.<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI the <partinfo>BBa_J61002</partinfo>-BBa_J231xx EcoRI-PstI fragment in <partinfo>pSB4C5</partinfo>, in order to transfer the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
<br />
Doubling time were evaluated for the described cultures:<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>doubling time [minutes]</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || <br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||<br />
|}<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced LuxI protein amounts that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformans could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluoresent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced</b>===<br />
<br />
'''Experimental implementation''' The new parts were, thus, characterized, measuring the HSL concentration released in the medium after a 6 hour growth of the cultures. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 sensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts contained in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in high copy plasmid]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced after 6 hours growth by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> is reported in Fig.9 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in low copy plasmid]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) beared on high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized here:<br />
<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan="2" align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|400px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|400px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|400px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|400px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]]<br />
*K_HSL is the HSL synthesys rate per cell, it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis to estimate the HSL synthesys rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double. It was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
|[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> ± <br> *<br />
| 8.94 10^-17 <br> ± <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> ± <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> ± <br> *<br />
| 7.53 10^-18 <br> ± <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> ± <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
'''Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center' colspan='2'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300030.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300028.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300029</partinfo><br> (wiki name: I16)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300029.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| 0.31 <br> ± <br> *<br />
| 1.17 10^-16 <br> ± <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> ± <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300025.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300026.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300027.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 700px; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two self-inducible devices (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were realized and characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell and an algorithm was proposed, in order to evaluate the O.D.start for every self-inducible device.<br />
<br />
In Figure, the O.D.start as a function of the strength of the promoter (RPU) controlling the Signal Molecule production is reported, both for <partinfo>BBa_K300010</partinfo> (Sender&Receiver device in HC plasmid) and for <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo> (Sender device in LC plasmid in combination with Receiver device in HC plasmid).<br />
<br />
{|<br />
|[[Image:pv_RPUvsOD_HCHC_M9.png|700px|thumb|center| O.D.start as a function of RPUs of the promoters controlling the Signal Molecule production for <partinfo>BBa_K300010</partinfo> in high copy number plasmid in M9 medium]]<br />
|[[Image:pv_RPUvsOD_HCLC_M9.png|700px|thumb|center| O.D.start as a function of RPUs of the promoters controlling the Signal Molucule production for <partinfo>BBa_K300010</partinfo> in low copy number plasmid used in combination with <partinfo>BBa_F2620</partinfo> in high copy plasmid in M9 medium]]<br />
|}<br />
<br />
A strong correlation between the promoter strength, previously measured in RPU, and the O.D.start is depicted by data. This is consistent with the expected behavior of these parts, since the HSL synthesis rate results stronger at the increase of promoter's strength.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div></div>Susannahttp://2010.igem.org/File:Pv_RPUvsOD_HCLC_M9.pngFile:Pv RPUvsOD HCLC M9.png2010-10-27T10:08:38Z<p>Susanna: </p>
<hr />
<div></div>Susannahttp://2010.igem.org/File:Pv_RPUvsOD_HCHC_M9.pngFile:Pv RPUvsOD HCHC M9.png2010-10-27T10:08:31Z<p>Susanna: </p>
<hr />
<div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewPartsTeam:UNIPV-Pavia/Parts/Characterization/NewParts2010-10-26T22:39:01Z<p>Susanna: /* BBa_K300010 - PoPS-based self-inducible device */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><td valign=top width="70%"><br />
<br />
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=<partinfo>BBa_K300000</partinfo> - BioBrick integrative base vector for ''E. coli''=<br />
<br />
==Materials and Methods==<br />
<br />
'''Plasmids and strains:''' the <partinfo>BBa_J72008</partinfo> helper plasmid was kindly given by Prof. JC Anderson (UC Berkeley). BW23474 (<partinfo>BBa_K300985</partinfo>), MC1061 (<partinfo>BBa_K300078</partinfo>) and MG1655 (<partinfo>BBa_V1000</partinfo>) E. coli strains and the pCP20 helper plasmid were purchased from the Coli Genetic Stock Center (Yale University).<br />
<br />
<br />
'''Verification primers:''' all the oligonucleotides were purchased from Primm (San Raffaele Biomedical Science Park, Milan, Italy). The P1 (<partinfo>BBa_K300975</partinfo>) and P4 (<partinfo>BBa_K300978</partinfo>) primers had already been used in [Anderson JC et al., 2010]. The P2 (<partinfo>BBa_K300976</partinfo>) and P3 (<partinfo>BBa_K300977</partinfo>) primers have been newly designed using ApE and Amplify 3X. P2 and P3 have been designed also considering the previously used verification primers P2 and P3 in the pG80ko integrative plasmid, described in [DeLoache W, 2009].<br />
<br />
The relative position of the P1, P2, P3 and P4 primers is shown in Fig.1:<br />
<br />
{|align=center<br />
|[[Image:relativeprimers.png|thumb|450px|center|Figure 1: Relative position of the verification primers. a) no integrants; b) single integrant and c) integrant with multiple tandem copies. P1/P2 and P3/P4 pairs give an amplicon when at least one copy of the vector is integrated in the Phi80 locus. P2/P3 pair show an amplicon only when multiple tandem copies occur.]]<br />
|}<br />
<br />
'''Competent cells preparation:''' all the ''E. coli'' strains were made competent following a slightly modified version of the protocol described in [Sambrook J et al., 1989]. Briefly, cells were grown to and OD600 of ~0.4-0.6, harvested (4000 rpm, 10 min, 4°C) and the supernatant discarded. Cells were resuspended in (30 ml for each 50 ml of initial culture) pre-chilled Mg-Ca buffer (80 mM MgCl2, 20 mM CaCl2), centrifuged as before and the supernatant discarded. Cells were resuspended in (2 ml for each 50 ml of initial culture) pre-chilled Ca buffer (100 mM CaCl2, 15% glycerol), aliquoted in 0.5 ml tubes and freezed immediately at -80°C. Test the transformation efficiency in Colony Forming Units (CFU)/ug of transformed DNA<br />
<br />
The Chloramphenicol concentration in plates was 34 ug/ml for the high copy plasmids, 12.5 ug/ml for the medium/low copy plasmids and 12.5 for the three control strains transformed with the R6K plasmid.<br />
<br />
<br />
'''Integration protocol:'''<br />
<br />
# Transform the <partinfo>BBa_J72008</partinfo> helper plasmid in the host strain (MC1061 or MG1655) and select transformants on Amp (50 ug/ml) plates under permissive conditions (30°C) overnight.<br />
# Inoculate a single colony in selective LB and let the culture grow at 30°C, 220 rpm. When the culture reaches the OD600 of 0.4-0.6 prepare chemically competent cells.<br />
# Transform the integrative vector with the desired insert in the BBa_J72008-containing strain and select co-transformants on Cm (34 ug/ml) plates under permissive conditions (30°C) overnight. At this temperature <partinfo>BBa_J72008</partinfo> can be replicated and so the pir protein product can be expressed in the cells. The pir product enables the propagation of the integrative vector by replicating the R6K origin.<br />
# Inoculate a single colony in 5 ml of LB + Cm at 12.5 ug/ml and incubate the culture at 37°C, 220 rpm overnight. At this temperature the <partinfo>BBa_J72008</partinfo> helper cannot be replicated and the Phi80 integrase is expressed by the remaining copies of the helper. The bacteria that are able to grow in this selective medium should be correct integrants because the integrative vector cannot be replicated by the pir product anymore.<br />
# Streak the culture on a Cm plate (at 12.5 ug/ml) and incubate it at 43°C overnight to ensure the loss of the helper plasmid. The bacteria that form colonies should be correct integrants without the <partinfo>BBa_J72008</partinfo> helper plasmid.<br />
<br />
Validate the loss of the helper plasmid by inoculating colonies in Cm (at 12.5 ug/ml) media and counterselecting them in Amp (at 50 ug/ml) media. Validate the correct integration position by performing colony PCR with primers P1/P2, P3/P4, P1/P4, P2,P3 and VF2/VR. Validate the phenotype (when possible).<br />
<br />
<br />
Expected amplicon length [bp] when the vector is integrated into the Phi80 locus:<br />
{|border=1<br />
|&nbsp;<br />
|'''No integrant'''<br />
|'''Single integrant'''<br />
|'''Multiple tandem integrants (>1)'''<br />
|-<br />
|'''VF2/VR'''<br />
|none<br />
|280 + insert length<br />
|280 + insert length<br />
|-<br />
|'''P1/P4'''<br />
|546<br />
|546 + insert length + 2171 (i.e. the BBa_K300000 length)<br />
|546 + insert length + 2171 (i.e. the BBa_K300000 length)<br />
|-<br />
|'''P1/P2'''<br />
|none<br />
|452<br />
|452<br />
|-<br />
|'''P3/P4'''<br />
|none<br />
|666<br />
|666<br />
|-<br />
|'''P2/P3'''<br />
|none<br />
|none<br />
|572<br />
|}<br />
<br />
<br />
'''Marker excision protocol:'''<br />
<br />
# Inoculate an integrant in selective LB medium and let it grow to OD600=0.4-0.6. Prepare chemically competent cells.<br />
# Transform the pCP20 helper plasmid in the competent strain and select transformants on Amp (100 ug/ml) plates under permissive conditions (30°C) overnight. At this temperature the pCP20 can be replicated. The pCP20 plasmid contains Amp and Cm resistance markers, a thermoinducible Flp recombinase expression system and a heat-sensitive replication origin. The permissive temperatures for the pCP20 propagation are the same as <partinfo>BBa_J72008</partinfo>.<br />
# Inoculate a single colony in 5 ml of LB without antibiotic and incubate the culture at 37°C, 220 rpm overnight. At this temperature the pCP20 helper cannot be replicated and the Flp recombinase is expressed by the remaining copies of the helper. The bacteria should loose the R6K origin and the Cm resistance upon FRT sites recombination, mediated by Flp.<br />
# Streak the culture on a LB plate and incubate it at 43°C overnight to ensure the loss of the helper plasmid. The bacteria that form colonies should be without the pCP20 helper plasmid.<br />
Validate the loss of the helper plasmid by inoculating colonies in Amp (at 100 ug/ml) media and validate the loss of the Cm resistance from the genome by inoculating colonies in Cm (at 12.5 ug/ml) media. Validate the correct length of the integrated part without Cm resistance and R6K origin by performing colony PCR with primers P1/P4 (which amplify the entire Phi80 locus) and VF2/VR (which amplify the integrated part). Validate the phenotype (when possible).<br />
<br />
<br />
'''Colony PCR:''' a single colony or 1 ul of culture was added to the Invitrogen Platinum Taq reaction mix and was heated at 94°C for 10 min. Then it was assayed with this cycle (X 35): 94°C 30 sec, 60°C (for VF2/VR) or 63°C (for the other primers) 30 sec, 72°C according to the amplicon expected length (1Kb/min). Then the reaction was kept at 72°C for 10 min and it was run on a 1% agarose gel with the GeneRuler 1Kb Plus DNA ladder (Fermentas).<br />
<br />
<br />
'''Fluorescence assays:''' integrants were inoculated in 1 ml of M9 + Cm (12.5 ug/ml) and grown at 37°C, 220 rpm overnight. The cultures were diluted 1:100 in 2 ml of selective M9 and let grow for about 4-6 hours under the same conditions as before. Three 200 ul aliquots for each culture were transferred to a 96-well microplate and assayed in the Infinite F200 microplate reader (Tecan) for about 20 hours with the following kinetic cycle: 37°C, 5 min sampling time, linear shaking 15 sec (amplitude=3), wait 5 sec, measure absorbance at 600nm, measure fluorescence with the proper filter (EX:nm/EM:540nm for GFP or EX:535nm/EM:620nm for RFP) with gain=70. The same protocol was followed for the MC1061 and the MG1655 non-integrant strains, which were grown in M9 without antibiotic.<br />
<br />
<br />
'''Data analysis:''' the absorbance measurements were normalized by subtracting the absorbance of the M9, while the fluorescence measurements were normalized by subtracting the fluorescence of the non-integrant strains over time. For each well, the S<sub>cell</sub> signal (proportional to the reporter protein synthesis rate per cell) was computed as (1/OD600*dXFP/dt), where OD600 is the normalized absorbance and XFP is the normalized fluorescence. The S<sub>cell</sub> signal was then averaged over time to obtain a single value for each well. Results are presented as the average S<sub>cell</sub> with their 95% confidence intervals of the mean.<br />
<br />
==Results==<br />
<br />
===Integration of the desired BioBrick part into the Phi80 genome locus===<br />
<br />
MC1061 and MG1655 were chosen as host strains for integration. <partinfo>BBa_K173001</partinfo> (constitutive strong promoter with GFPmut3) and the EcoRI-PstI fragment of <partinfo>BBa_J61002</partinfo>-<partinfo>BBa_J23101</partinfo> (here called PconRFP - constitutive strong promoter with RFP) were chosen as two proof of concept BioBrick parts to test the integration capability of the <partinfo>BBa_K300000</partinfo> vector in the Phi80 genome locus of these strains. For this reason, <partinfo>BBa_K173001</partinfo> and PconRFP were ligated in <partinfo>BBa_K300000</partinfo> (digested with EcoRI-PstI) and propagated using BW23474.<br />
The integration protocol was performed as described in the Materials and Methods section for 4 different combination:<br />
<br />
{|border=1<br />
|'''Integrant name'''<br />
|'''Strain'''<br />
|'''Insert of <partinfo>BBa_K300000</partinfo><br />
|-<br />
|MC-GFP<br />
|MC1061<br />
|<partinfo>BBa_K173001</partinfo><br />
|-<br />
|MC-RFP<br />
|MC1061<br />
|PconRFP<br />
|-<br />
|MG-GFP<br />
|MG1655<br />
|<partinfo>BBa_K173001</partinfo><br />
|-<br />
|MG-RFP<br />
|MG1655<br />
|PconRFP<br />
|}<br />
<br />
Three colonies grown after the overnight incubation at 43°C (step 5 of integration protocol) were analyzed for each plate. These 12 clones were called: MC-GFP-A,B,C , MC-RFP-A,B,C , MG-GFP-A,B,C and MG-RFP-A,B,C.<br />
<br />
<br />
'''Validation of the loss of BBa_J72008:''' all the picked colonies did not grow in Amp (50 ug/ml) media, thus validating that <partinfo>BBa_J72008</partinfo> Amp-resistant helper had been actually cured from the cells. However, one of these 12 clones (MG-GFP-A) also failed to grow in Cm (12.5 ug/ml) liquid media, probably because of a mistake in its inoculation. We decided not to consider this clone and to continue with 11 clones.<br />
<br />
<br />
'''Validation of the actual integration site:''' colony PCR was performed for all the 11 clones, using MC1061 and MG1655 without integrants as negative controls. Primer pairs P1/P2 and P3/P4 were used to validate the presence of the integrative vector in the Phi80 genomic locus, while the primer pair P2/P3 was used to validate the presence of multiple tandem integrants (see Fig.1 in Materials and Methods).<br />
<br />
<br />
{|align=center<br />
|[[Image:pv_P1P2.png|thumb|450px|center|Figure 2: colony PCR with P1/P2 on all the 11 integrant clones. The blank is the reaction mix without bacteria. Expected amplicon for correct integrants: 452 bp.]]<br />
|}<br />
<br />
{|align=center<br />
|[[Image:pv_P3P4.png|thumb|450px|center|Figure 3: colony PCR with P3/P4 on all the 11 integrant clones. The blank is the reaction mix without bacteria. Expected amplicon for correct integrants: 666 bp.]]<br />
|}<br />
<br />
{|align=center<br />
|[[Image:pv_P2P3.png|thumb|450px|center|Figure 4: colony PCR with P2/P3 on all the 11 integrant clones. The blank is the reaction mix without bacteria. The lanes with the amplicon were expected to come from bacteria with multiple tandem integrants. Expected amplicon for multiple integrants: 572 bp.]]<br />
|}<br />
<br />
<br />
PCR results with primers P1/P2 and P3/P4 showed that each clone had the correct integrant in the correct genomic position (see Materials and Methods for a list of the expected amplicon lengths). Negative controls showed no amplicons with primers P1/P2 as expected, but showed an unexpected band with P3/P4. The reason of the presence of this band was not further investigated and the results with this primer pair cannot be a useful tool for future analysis. Anyway, the P1/P2 primer pair can be sufficient to successfully validate the presence of the DNA of interest in the Phi80 genomic locus.<br />
<br />
PCR results with primers P2/P3 showed that two clones (MC-GFP-B and MC-GFP-C) were single integrants, while all the other clones were multiple tandem integrants (i.e. the Phi80 locus contained more than one copy of the DNA of interest). Negative controls showed no amplicons, as expected.<br />
<br />
<br />
'''Validation of the integrants phenotype:''' all the 11 clones were assayed as described in the Materials and Methods section. Unfortunately, the green fluorescent clones (MC-GFP-A,B,C and MG-GFP-B,C) did not show appreciable differences when compared to negative controls, most probably because the autofluorescence of the cells was too high and hid the GFP signal. For this reason, GFP clones were not considered for further analysis. Other instruments should be used to detect the GFP signal.<br />
<br />
On the other hand, RFP clones (MC-RFP-A,B,C and MG-RFP-A,B,C) all showed a higher fluorescence than the negative controls (see Fig.5). As Fig.5 show, the fluorescence of the three MG-RFP had a higher variability between clones when compared to the three MC-RFP. However, the clones were not necessarily expected to behave in the same way because all of them were multiple tandem integrants and the copy number of the PconRFP construct could be arbitrary.<br />
<br />
<br />
{|align=center<br />
|[[Image:pv_phenotypeRFPbefore.png|thumb|450px|center|Figure 5: relative GFP synthesis rate for all the RFP expressing clones.]]<br />
|}<br />
<br />
<br />
===Chloramphenicol resistance marker excision===<br />
<br />
The marker excision was performed on two of the previously validated integrant strains: MC-RFP-A and MG-RFP-A (even if they were multiple tandem integrants).<br />
<br />
The marker excision protocol was performed as described in the Materials and Methods section for both strains, here named:<br />
<br />
{|border=1<br />
|'''Original name'''<br />
|'''Name after marker excision'''<br />
|-<br />
|MC-RFP<br />
|MC-RFPflip<br />
|-<br />
|MG-RFP<br />
|MG-RFPflip<br />
|}<br />
<br />
<br />
Three colonies grown after the overnight incubation at 43°C (step 4 of marker excision protocol) were analyzed for each plate. These 6 clones were called MC-RFPflip-A,B,C and MG-FRPflip-A,B,C.<br />
<br />
<br />
'''Validation of the loss of pCP20 and the resistance marker:''' all the 6 picked colonies failed to grow on both Amp (100 ug/ml) media and Cm (12.5 ug/ml) media. They could only grow in LB without antibiotics, thus validating that the pCP20 helper had been actually cured and the R6K-CmR DNA containing the Chloramphenicol selection marker had been actually eliminated.<br />
<br />
<br />
'''Validation of the length of the integrated part:''' colony PCR was performed for all the 6 clones, using MC1061 and MG1655 without integrants as negative controls. Primer pairs VF2/VR and P1/P4 were used to validate if the ''passenger'' of interest was still present in the genome and the length of the entire Phi80 locus respectively after the marker excision.<br />
<br />
{|align=center<br />
|[[Image:pv_VF2VRintegrants.png|thumb|450px|center|Figure 6: colony PCR with VF2/VR on all the 6 flipped clones. The blank is the reaction mix without bacteria. Expected amplicon for correct insert: 1.2 Kb.]]<br />
|}<br />
<br />
{|align=center<br />
|[[Image:pv_P1P4integrants.png|thumb|450px|center|Figure 7: colony PCR with P1/P4 on all the 6 flipped clones. The blank is the reaction mix without bacteria. Expected amplicon for correct construct in the correct position: 2.3 Kb. Expected amplicon for the non-integrant strain MG1655: 546 bp.]]<br />
|}<br />
<br />
<br />
PCR results with primers VF2/VR showed that all the 6 clones still contain the ''passenger'' of interest, i.e. PconRFP, in the genome after the marker excision. The reaction blank, the MG1655 strain (neg control) and also the other samples showed some extra bands, but the ~1.2Kb amplicons of MC-RFP-A,B,C and MG-RFP-A,B,C had the correct length and was much brighter than the other bands.<br />
<br />
<br />
PCR results with primers P1/P4 (Fig.7) showed that an amplicon of ~2.3Kb was present in all but one screened clones, while the MG1655 negative control showed the expected 546bp length for a non-integrant. MC-RFPflip-C did not show the P1-P4 amplicon because the reaction failed: the tube was damaged and the reaction mix was completely evaporated at the end of the PCR program. For this reason, a PCR was performed again on this clone (Fig.8).<br />
<br />
The ~2.3Kb amplicon was consistent with a single integrant of <partinfo>BBa_K300000</partinfo>-PconRFP without the R6K-CmR DNA fragment, thus validating the successful excision of the FRT-flanked DNA fragment containing R6K-CmR and confirming that PconRFP was still present in the correct locus in single copy.<br />
<br />
<br />
{|align=center<br />
|[[Image:pv_P1P4singlecloneintegrant.png|thumb|300px|center|Figure 8: colony PCR with P1/P4 on MC-RFPflip-C clone. The blank is the reaction mix without bacteria. Expected amplicon for correct construct in the correct position: 2.3 Kb.]]<br />
|}<br />
<br />
<br />
These results showed that, even if the clones were multiple tandem integrants, they became single integrants after marker excision. This is because the Flp recombinase mediated the recombination of all the FRT sites of the multiple integrants until only a single FRT site was present in the Phi80 locus, thus leaving only the single integrant of interest without the selection marker in the genome.<br />
<br />
<br />
'''Validation of the marker-less phenotype:'''all the 6 clones were assayed as described in the Materials and Methods section. They all showed a low variability and their fluorescence was lower than their two ''parents'', i.e. MC-RFP-A for the MC1061 strains and MG-RFP-A for the MG1655 strains (see Fig.9). This result is consistent with the copy number of the PconRFP construct in the clones, in fact both MC-RFP-A and MG-RFP-A were multiple tandem integrants, while MC-RFPflip-A,B,C and MG-RFPflip-A,B,C were single integrants, as described above.<br />
<br />
All the MG-RFPflip showed a very low relative RFP synthesis rate when compared to the other strains, but the signal is systematically grater than the fluorescence of the negative control, thus validating the phenotype for the MG1655 strain. MC-RFPflip-A,B,C showed a higher fluorescence than MG-RFPflip-A,B,C.<br />
<br />
In conclusion, it has been demonstrated that, even after the marker excision process, the phenotype of the engineered cells is maintained.<br />
<br />
{|align=center<br />
|[[Image:pv_phenotypeRFP.png|thumb|450px|center|Figure 9: relative RFP synthesis rate for all the RFP-expressing clones after marker excision. In this figure, the bars corresponding to the fluorescence of the clones before marker excision is also reported to facilitate the comparison between them. Note that all the three ''flip'' clones are derived from MC-RFP-A for the MC1061 clones and from MG-RFP-A for the MG1655 clones.]]<br />
|}<br />
<br />
==Discussion==<br />
<br />
A novel integrative vector for ''E. coli'' has been successfully designed, constructed and used to integrate two proof of concept protein expression systems in two commonly used E. coli strains.<br />
<br />
The results showed that the vector is fully functional and can integrate into the correct targeted locus of the host chromosome through the Phi80 site-specific recombination system by using <partinfo>BBa_J72008</partinfo>, an existing BioBrick helper plasmid from the Registry. In most cases, the integration occurs in tandem copies, probably because of the too high Chloramphenicol concentration used during the selection of integrants, which forces multiple integration of Cm-resistant constructs. This concentration was the same used during the pSC101 low copy plasmid (~5 copies per cell) selection. In some cases, it is desirable to have a single copy of the desired BioBrick in the genome, for example when the gene dosage is important. In [Haldimann A and Wanner BL, 2001] the usage of Chloramphenicol at 6 ug/ml yielded a very high percentage of single integrants. However, when tested in our lab, the MG1655 strain could survive on LB plates with Cm at 6 ug/ml and also at 8 ug/ml. For this reason a higher concentration of Cm was chosen for selection. Further studies should investigate the optimal antibiotic concentration to yield the highest single integrants percentage as possible.<br />
<br />
<br />
The Flp/FRT mediated excision of the R6K and, most importantly, of the Cm resistance marker also worked by using the pCP20 helper plasmid. The estimated efficiency of this process was 100%. In addition, multiple tandem integrants became single integrants after the marker excision. This is because the Flp recombinase mediated the recombination of all the FRT sites of the multiple integrants until only a single FRT site was present in the Phi80 locus. The marker excision is a powerful tool to engineer microbial strains for industrial protein manufacturing because the engineered organism should not carry unsafe antibiotic resistances that may be diffused in the environment.<br />
<br />
<br />
The fluorescence phenotype confirmed the correct integration into the ''E. coli'' chromosome. As expected, in general multiple integrants showed a higher fluorescence than the single integrants.<br />
<br />
<br />
The BioBrick compatibility and the vector modularity give the possibility to the scientific community to stably engineer novel biological functions in ''E. coli'' with a very easy and user friendly methodology. A user’s handbook about the vector usage is shared in the Registry, as well as the users experiences and the compatibility information.<br />
<br />
<br />
<br />
=<partinfo>BBa_K300001</partinfo> - BioBrick integrative base vector for ''S. cerevisiae''=<br />
The integration capability of this vector has been tested in S288C ''S. cerevisiae'' strain (<partinfo>BBa_K300979</partinfo>). Here is reported the followed protocol and the obtained results.<br />
<br />
<br />
'''Protocol:'''<br />
<br />
*S288C strain (Open Biosystems) was inoculated in 5 ml of YPD from a long term 15% glycerol stock and grown for 24h (30°C, 200rpm).<br />
*The culture was diluted 1:10 in 50 ml of pre-warmed YPD in a 250 ml flask and was grown for additional 4 hours under the same conditions as before.<br />
*Cells were pelleted (4000 rpm, 5 min) and resuspended in 25 ml of deionized water.<br />
*Cells were pelleted (4000 rpm, 5 min), the supernatant was discarded and the pellet was resuspended in 1 ml of deionized water and transferred into a 1.5 ml tube.<br />
*Cells were pelleted (4000 rpm, 30 sec), the supernatant was discarded and the pellet was resuspended in deionized water to a final volume of 1 ml (vortex mix vigorously).<br />
*Three 100 ul aliquots were transferred into 1.5 ml tubes, while the remaining 600 ul of cells were not used in this protocol.<br />
*The three tubes were centrifuged (4000 rpm, 30 sec) and the supernatant discarded.<br />
*Each of the three pellets were resuspended (vortex mix vigorously) in 360 ul of transformation mix (240 ul of PEG 3350 50% w/v, 36 ul of LiAc 1.0 M, boiled salmon sperm DNA, 34 ul of linearized plasmid DNA plus water). The salmon sperm DNA was boiled for 5 min and pre-chilled before adding it in the transformation mix. The plasmid DNA was previously digested with SbfI (Fermentas), purified with the NucleoSpin Extract II kit (MN) and quantified with the NanoDrop in order to add 1 ug of DNA to the transformation mix.<br />
*The tubes were heated at 42°C for 40 min.<br />
*Cells were pelleted (4000 rpm, 30 sec), the supernatant was removed by pipetting and the pellet was gently resuspended in 1 ml of deionized water.<br />
*Cells were pelleted (4000 rpm, 30 sec), the supernatant was discarded, the pellet was resuspended in 1 ml of YPD and incubated at 30°C, 200 rpm for 3 hours.<br />
*Cells were pelleted (4000 rpm, 30 sec), resuspended in 200 ul of YPD and plated on a YPD agar plate with G418 antibiotic at 200 ug/ml.<br />
*The plates were incubated at 30°C for about 3 days until colonies appeared.<br />
<br />
<br />
The integration efficiency was estimated as the colony forming units (CFUs) yielded for each ug of DNA.<br />
<br />
<br />
Protocol references:<br />
<br />
[1] http://openwetware.org/wiki/High_Efficiency_Transformation<br />
<br />
[2] Guldener U, Heck S, Fiedler T, Beinhauer J, Hegemann JH (1996), A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Research, Vol. 24, No. 13 2519–2524.<br />
<br />
<br />
'''Results:'''<br />
<br />
The transformed inserts and their integration efficiency in S288C are listed here:<br />
<br />
{|border=1<br />
|'''SbfI-digested plasmid'''<br />
|'''ug of transformed DNA'''<br />
|'''# of colonies'''<br />
|'''Estimated integration efficiency [CFU/ug]'''<br />
|-<br />
|<partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300006</partinfo><br />
|1<br />
|1700<br />
|1.7*10^3<br />
|-<br />
|<partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300007</partinfo><br />
|1<br />
|6500<br />
|6.5*10^3<br />
|-<br />
|no DNA<br />
|0<br />
|0<br />
|0<br />
|}<br />
<br />
<br />
These results suggest that the integrative vector actually works and that the selection marker is highly specific (no colonies appeared on the "no DNA" plate).<br />
<br />
The correct phenotype of the S288C bearing these parts has still to be validated (by mOrange fluorescence measurement for the <partinfo>BBa_K300007</partinfo> part), as well as the actual integration position (by PCR).<br />
<br />
=<partinfo>BBa_K300004</partinfo> - Engineered pH-inducible intein (codon optimized for E. coli) - internal domain=<br />
=<partinfo>BBa_K300010</partinfo> - PoPS-based self-inducible device=<br />
<br />
The PoPS-based self-inducible device should be assembled downstream of a promoter and upstream of the gene of interest to obtain a device able to start the production of the target protein at a pre-determined user-defined culture density.<br />
''LuxI'' encodes a protein responsible for the synthesis of 3-oxo-C6-homoserine-lactone (3OC6HSL or simply HSL) and its production should be regulated by a different promoter, depending on the deisred O.D.start.<br />
''luxR'' encodes a protein capable to bind the HSL and is constitutively expressed under the control of ''pTetR'' promoter (<partinfo>BBa_R0040</partinfo>).<br />
<br />
The features of this device have been evaluated in many different experimental conditions: <br />
* varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
* varying the copy number of vectors bearing both Sender and Receiver circuits<br />
* varying the growth medium (LB or M9) <br />
<br />
The results obtained are reported in the sections below.<br />
<br />
=<partinfo>BBa_K300093</partinfo>, <partinfo>BBa_K300094</partinfo>, <partinfo>BBa_K300097</partinfo>, <partinfo>BBa_K300095</partinfo> and <partinfo>BBa_K300084</partinfo> - Phasin and Intein-based TAGs for protein purification=<br />
<br />
These parts are built by assembling Phasins (<partinfo>BBa_K300002</partinfo> and <partinfo>BBa_K300003</partinfo>) that are able to bind to PolyHydroxyAlkanoates (PHA)<br />
granules and Intein (<partinfo>BBa_K300004</partinfo>), a sequence capable of self-exciding from a precursor protein through a process known as self-splicing. In addition a flexible linker sequence (<partinfo>BBa_K105012</partinfo>) has been used to connect these parts in order to facilitate the binding and folding of the tag and the target protein of interest. Thanks to Phasin and Intein properties these parts can be used as high-specific TAGs for low-cost protein purification.<br />
<br />
At the moment were were not able to test Intein efficiency, but we could check if they affected the right folding of the target protein: we achieved this goal through the Silver Standard Assembly by using the GFP (<partinfo>BBa_K300005</partinfo>).<br />
<br />
These parts has been characterized respectively through:<br />
*pTet costitutive promoter devices:<br />
**<partinfo>BBa_K300088</partinfo><br />
**<partinfo>BBa_K300090</partinfo><br />
**<partinfo>BBa_K300099</partinfo><br />
*3OC6HSL inducible devices:<br />
**<partinfo>BBa_K300091</partinfo><br />
**<partinfo>BBa_K300092</partinfo><br />
and compared to a positive (<partinfo>BBa_K173000</partinfo>) and negative (<partinfo>BBa_B0031</partinfo>) control.<br />
<br />
==pTet costitutive promoter devices==<br />
===Methods===<br />
Inoculum (into 5 ml LB+Amp) from glycerol stock of:<br />
*<partinfo>BBa_K300088</partinfo><br />
*<partinfo>BBa_K300090</partinfo><br />
*<partinfo>BBa_K300099</partinfo><br />
*<partinfo>BBa_K173000</partinfo> (positive control)<br />
*<partinfo>BBa_B0031</partinfo> (negative control)<br />
They were let grow ON at +37°C, 220 rpm.<br />
<br />
The following day cultures were diluted 1:100 and let grow again for about five hours at +37°C, 220 rpm.<br />
<br />
Optical density (O.D.) of each cell culture was than measured with TECAN Infinte F200. Samples were diluted in order to obtain the same O.D. equal to 0,02.<br />
<br />
Than we performed a 21 hours' experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200. Each value shown is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted; cultures were shaken for 15 seconds every five minutes.<br />
<br />
===Results===<br />
<br />
{|align="center"<br />
|[[Image:UNIPV10_pTET_newP_ASB.png|thumb|300px|Raw growth curve]] || [[Image:UNIPV10_pTET_newP_GFP.png|thumb|300px|Raw GFP curve]]<br />
|}<br />
<table align="center"><br />
<tr><br />
<td><br />
[[Image:UNIPV10_pTET_newP_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<table border="1" align="center"><br />
<tr align="center"><br />
<th>Culture</th><th>Doubling time [min.]</th><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K173000</partinfo></td><td>77</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300088</partinfo></td><td>75</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300090</partinfo></td><td>76</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300099</partinfo></td><td>76</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_B0031</partinfo></td><td>69</td><br />
</tr><br />
</table><br />
<br />
===Discussion===<br />
All cell cultures showed a similar growth curve and doubling time was computed as described [[Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation|here]] in order to have informations about the burden due to synthesis of such fusion proteins. It's possible to see that all doubling time are very similar; it's possible to assert that the expression of these BioBrick parts doesn't cause abnormal stress to the cells.<br />
<br />
In GFP curve it's possible to appreciate that in <partinfo>BBa_K300088</partinfo>, <partinfo>BBa_K300090</partinfo>, <partinfo>BBa_K300099</partinfo> GFP accumulation it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. These results show the right folding of the green fluorescent protein assembled downstream of the genetic circuit.<br />
<br />
The mean protein synthesis rate was also computed over the growth exponential phase, showing again an appreciable GFP production rate that is about a half of the positive control.<br />
<br />
==3OC6HSL inducible devices==<br />
===Methods===<br />
Inoculum (into 5 ml LB+Amp) from glycerol stock of:<br />
*<partinfo>BBa_K300091</partinfo><br />
*<partinfo>BBa_K300092</partinfo><br />
*<partinfo>BBa_K173000</partinfo> (positive control)<br />
*<partinfo>BBa_B0031</partinfo> (negative control)<br />
They were let grow ON at +37°C, 220 rpm.<br />
<br />
The following day cultures were diluted 1:100 and let grow again for about five hours at +37°C, 220 rpm.<br />
<br />
Optical density (O.D.) of each cell culture was than measured with TECAN Infinte F200. Samples were diluted in order to obtain the same O.D. equal to 0,02.<br />
<br />
Than we performed a 21 hours' experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200. <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> circuits were induced 100nM with HSL directly into multiplate well. Each value shown is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted; cultures were shaken for 15 seconds every five minutes.<br />
<br />
===Results===<br />
<table align="center"><br />
<tr><br />
<td>[[Image:UNIPV10_HSL_c_ASB.png|thumb|300px|Raw growth curve]]</td><br />
<td>[[Image:UNIPV10_HSL_c_GFP.png|thumb|300px|Raw GFP curve]]</td><br />
</tr><br />
</table><br />
<table align="center"><br />
<tr><br />
<td>[[Image:UNIPV10_HSL_c_BAR.png|thumb|300px|Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)]]</td><br />
</tr><br />
</table><br />
<br />
<table border="1" align="center"><br />
<tr align="center"><br />
<th>Culture</th><th>Doubling time [min.]</th><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K173000</partinfo></td><td>75</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300091</partinfo><br/>induced</td><td>121</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300091</partinfo><br/>not induced</td><td>74</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300092</partinfo><br/>induced</td><td>123</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_K300092</partinfo><br/>not induced</td><td>72</td><br />
</tr><br />
<tr align="center"><br />
<td><partinfo>BBa_B0031</partinfo></td><td>74</td><br />
</tr><br />
</table><br />
===Discussion===<br />
All cell cultures showed a similar growth curve and doubling time was computed as described [[Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation|here]] in order to have informations about the burden due to synthesis of such fusion proteins. It's possible to see that all doubling time are very similar except for induced cultures. In this case doubling time is much higher than posite control and not induced cultures; so it's possible to assert that in this case there's a kind of metabolic burden higher than in the others, maybe because of the inducible system.<br />
<br />
In GFP curve it's possible to appreciate that in induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> GFP accumulation profile it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. On the other hand not induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> show a profile very similar to the last one. These results show the right folding of the green fluorescent protein assembled downstream of the genetic circuit and that the inducible system works as expected.<br />
<br />
The mean protein synthesis rate was also computed over the growth exponential phase, showing again an GFP production rate that is different from negative control. Not induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> show a low GFP synthesis rate maybe due to 3OC6HSL inducible circuit leakage activity.<br />
<br />
</td><br />
</tr></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAutoTeam:UNIPV-Pavia/Project/PromotoriAuto2010-10-26T22:29:35Z<p>Susanna: /* Results */</p>
<hr />
<div>__NOTOC__<br />
{{UNIPV-Pavia/header}} <br />
<br />
<br />
<table width="100%" border="0" align="center"><br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td align="justify" valign="top" style="padding:20px"><br />
<p align="center" valign="top"><br><br />
<font size="5" face="Cataneo BT" align="center" valign="top">'''''ProteInProgress: a cellular assembly line for protein manufacturing'''''</font><br />
</p><br />
<hr><br />
<br><br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/motivation"><br />
<img src="https://static.igem.org/mediawiki/2010/b/be/UNIPV_Pavia_motivation2_BN.jpg" width="75px" height="75px" alt="Motivation" title="Motivation"/><br />
</a><br />
</html></td><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/solution"><br />
<img src="https://static.igem.org/mediawiki/2010/c/c8/UNIPV_Pavia_solution2_BN.jpg" width="75px" height="75px" alt="Solutions" title="Solutions"/></a><br />
</html></td><br />
<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/results"><br />
<img src="https://static.igem.org/mediawiki/2010/7/77/UNIPV_Pavia_result.jpg" width="75px" height="75px" alt="Implementation & Results" title="Implementation & Results"/><br />
</a><br />
</html></td><br />
<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/references"><br />
<img src="https://static.igem.org/mediawiki/2010/6/61/UNIPV_Paviareferences_BN.jpg" width="75px" height="75px" alt="References" title="References"/></a><br />
</html></td><br />
</tr><br />
</table><br />
<br />
<html><a name="indice"/></a></html><br />
<br />
<p align="center" valign="top"><br><br />
<font size="5" align="center" valign="top"><b>Implementation and Results: Self-Inducible promoters</b></font><br />
</p><br />
<br><br />
<hr><br />
<br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAuto"><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/PromotoriAuto|Self-inducible promoters]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntColi"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/UNIPV_Pavia_Icona_intColi.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntColi|Integrative standard vector for E. coli]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntYeast"><br />
<img src="https://static.igem.org/mediawiki/2010/0/03/UNIPV_Pavia_Icona_IntYeast.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntYeast|Integrative standard vector for yeast]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/FasinaInteina"><br />
<img src="https://static.igem.org/mediawiki/2010/7/74/UNIPV_Pavia_Icona_Bioplastica.GIF" width="75px" height="75px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/FasinaInteina|Purification of proteins]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/><br />
</html><br />
<br />
=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein production</b>===<br />
<br />
'''Experimental implementation:''' <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:<br />
<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>Strength (a.u.)<br>reported in the Registry</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 2547<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||1791<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||623<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||1185<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||844<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||256<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||396<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||1429<br />
|}<br />
<br />
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]], using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:<br />
*high copy number plasmids and LB;<br />
*high copy number plasmids and M9;<br />
*low copy number plasmids and M9.<br />
It was not possible to evaluate promoters activities in low copy number plasmids and LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2]] and data were analyzed as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]]; <br />
<br />
'''Results''': results are shown here.<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI the <partinfo>BBa_J61002</partinfo>-BBa_J231xx EcoRI-PstI fragment in <partinfo>pSB4C5</partinfo>, in order to transfer the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
<br />
Doubling time were evaluated for the described cultures:<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>doubling time [minutes]</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || <br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||<br />
|}<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced LuxI protein amounts that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformans could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluoresent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced</b>===<br />
<br />
'''Experimental implementation''' The new parts were, thus, characterized, measuring the HSL concentration released in the medium after a 6 hour growth of the cultures. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 sensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts contained in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in high copy plasmid]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced after 6 hours growth by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> is reported in Fig.9 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in low copy plasmid]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) beared on high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized here:<br />
<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan="2" align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|400px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|400px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|400px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|400px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]]<br />
*K_HSL is the HSL synthesys rate per cell, it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis to estimate the HSL synthesys rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double. It was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
|[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> ± <br> *<br />
| 8.94 10^-17 <br> ± <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> ± <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> ± <br> *<br />
| 7.53 10^-18 <br> ± <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> ± <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
'''Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center' colspan='2'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300030.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300028.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300029</partinfo><br> (wiki name: I16)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300029.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| 0.31 <br> ± <br> *<br />
| 1.17 10^-16 <br> ± <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> ± <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300025.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300026.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300027.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
<br />
<div id="box" style="width: 700px; margin-left: 137px; padding: 5px; border: 1px solid #000; background-color: #f6f6f6;"><br />
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: black; padding: 5px;"><br />
LEGEND OF TABLES:<br />
</div><br />
<div id="legend" style="font-weight: normal; font-size: small; color: black; padding: 5px;"><br />
''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
</div><br />
</div><br />
<br />
'''Discussion''': two self-inducible devicec (<partinfo>BBa_K300010</partinfo> and <partinfo>BBa_K300009</partinfo>/<partinfo>BBa_F2620</partinfo>) were realized and characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell. <br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div></div>Susannahttp://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAutoTeam:UNIPV-Pavia/Project/PromotoriAuto2010-10-26T22:00:04Z<p>Susanna: /* Results */</p>
<hr />
<div>__NOTOC__<br />
{{UNIPV-Pavia/header}} <br />
<br />
<br />
<table width="100%" border="0" align="center"><br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td align="justify" valign="top" style="padding:20px"><br />
<p align="center" valign="top"><br><br />
<font size="5" face="Cataneo BT" align="center" valign="top">'''''ProteInProgress: a cellular assembly line for protein manufacturing'''''</font><br />
</p><br />
<hr><br />
<br><br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/motivation"><br />
<img src="https://static.igem.org/mediawiki/2010/b/be/UNIPV_Pavia_motivation2_BN.jpg" width="75px" height="75px" alt="Motivation" title="Motivation"/><br />
</a><br />
</html></td><br />
<td align="center"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/solution"><br />
<img src="https://static.igem.org/mediawiki/2010/c/c8/UNIPV_Pavia_solution2_BN.jpg" width="75px" height="75px" alt="Solutions" title="Solutions"/></a><br />
</html></td><br />
<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/results"><br />
<img src="https://static.igem.org/mediawiki/2010/7/77/UNIPV_Pavia_result.jpg" width="75px" height="75px" alt="Implementation & Results" title="Implementation & Results"/><br />
</a><br />
</html></td><br />
<td align="center"><br><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/references"><br />
<img src="https://static.igem.org/mediawiki/2010/6/61/UNIPV_Paviareferences_BN.jpg" width="75px" height="75px" alt="References" title="References"/></a><br />
</html></td><br />
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</table><br />
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<html><a name="indice"/></a></html><br />
<br />
<p align="center" valign="top"><br><br />
<font size="5" align="center" valign="top"><b>Implementation and Results: Self-Inducible promoters</b></font><br />
</p><br />
<br><br />
<hr><br />
<br />
<table align="center" border="0" width="80%"><br />
<tr><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/PromotoriAuto"><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/PromotoriAuto|Self-inducible promoters]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntColi"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/UNIPV_Pavia_Icona_intColi.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntColi|Integrative standard vector for E. coli]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/IntYeast"><br />
<img src="https://static.igem.org/mediawiki/2010/0/03/UNIPV_Pavia_Icona_IntYeast.GIF" width="55px" height="70px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/IntYeast|Integrative standard vector for yeast]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project/FasinaInteina"><br />
<img src="https://static.igem.org/mediawiki/2010/7/74/UNIPV_Pavia_Icona_Bioplastica.GIF" width="75px" height="75px"/></a><br />
</html><br />
<br><br />
[[Team:UNIPV-Pavia/Project/FasinaInteina|Purification of proteins]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/><br />
</html><br />
<br />
=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein production</b>===<br />
<br />
'''Experimental implementation:''' <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection]; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:<br />
<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>Strength (a.u.)<br>reported in the Registry</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || 2547<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||1791<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||623<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||1185<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||844<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||256<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||396<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||1429<br />
|}<br />
<br />
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]], using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:<br />
*high copy number plasmids and LB;<br />
*high copy number plasmids and M9;<br />
*low copy number plasmids and M9.<br />
It was not possible to evaluate promoters activities in low copy number plasmids and LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2|Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2]] and data were analyzed as reported in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for RPU evaluation|Data analysis for RPU evaluation]]; <br />
<br />
'''Results''': results are shown here.<br />
<br />
{| align='center'<br />
|[[Image:pv_RPU_HC_LB.png|330px|thumb|center|Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>) ]]||[[Image:pv_RPU_HC_M9.png|330px|thumb|center|Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (<partinfo>BBa_J61002</partinfo>)]]<br />
|}<br />
{| align='center'<br />
|[[Image:pv_RPU_LC_M9.png|330px|thumb|center|Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>). These plasmids were constructed by assembling the EcoRI-PstI the <partinfo>BBa_J61002</partinfo>-BBa_J231xx EcoRI-PstI fragment in <partinfo>pSB4C5</partinfo>, in order to transfer the RBS-RFP-TT expression construct from <partinfo>BBa_J61002</partinfo> to <partinfo>pSB4C5</partinfo>.]]<br />
|}<br />
<br />
Doubling time were evaluated for the described cultures:<br />
{| align='center' border='1'<br />
|<b>Promoter</b> || <b>doubling time [minutes]</b><br />
|-<br />
|<partinfo>BBa_J23100</partinfo> || <br />
|-<br />
|<partinfo>BBa_J23101</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>||<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>||<br />
|}<br />
<br />
'''Discussion''': we observed that the ranking previously documented in the Registry is not valid in all the conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick''' ||'''Description'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300028</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300029</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300025</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|-<br />
| <partinfo>BBa_K300026</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|-<br />
| <partinfo>BBa_K300027</partinfo>|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|-<br />
| <partinfo>BBa_K300017</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23118<br />
|-<br />
| <partinfo>BBa_K300014</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23110<br />
|-<br />
| <partinfo>BBa_K300015</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23114<br />
|-<br />
| <partinfo>BBa_K300016</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23116<br />
|-<br />
| <partinfo>BBa_K300012</partinfo>|| [[Image:pv_SignalGeneratorSensorDevice.png|300px]]<br>J23105<br />
|} <br />
<br />
Some of the promoters could not be cloned upstream of these devices because they produced LuxI protein amounts that give a high metabolic burden for ''E. coli'', so it was not possible to study all the combinations as transformans could not be obtained in some cases.<br />
For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluoresent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density.<br />
<br />
===<b>Quantification of the HSL produced</b>===<br />
<br />
'''Experimental implementation''' The new parts were, thus, characterized, measuring the HSL concentration released in the medium after a 6 hour growth of the cultures. All the details are available in [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for 3OC6-HSL quantification by means of BBa_T9002 sensor - Protocol #3|this section]].<br />
<br />
<partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced.<br />
<br />
'''Results''' The amount of 3OC6-HSL produced after a 6 hours growth by ''E. coli'' DH5alpha bearing the parts contained in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|Figure 8 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in high copy plasmid]]<br />
|}<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.7 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.04 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.09 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || 0.002 uM<br />
|}<br />
<br />
The amount of 3OC6-HSL produced after 6 hours growth by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> is reported in Fig.9 and in the table:<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|Figure 9 - <partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced in low copy plasmid]]<br />
|}<br />
<br />
<br />
{| border='1' align='center'<br />
| ''BioBrick'' || ''Wiki name''|| E. coli ''strain'' || [HSL]<br />
|-<br />
| <partinfo>BBa_K300030</partinfo> || I14|| DH5alpha || 0.005 uM<br />
|-<br />
| <partinfo>BBa_K300028</partinfo> || I15|| DH5alpha || 0.002 uM<br />
|-<br />
| <partinfo>BBa_K300029</partinfo> || I16|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300025</partinfo> || I17|| DH5alpha || 0.003 uM<br />
|-<br />
| <partinfo>BBa_K300026</partinfo> || I18|| DH5alpha || not detected<br />
|-<br />
| <partinfo>BBa_K300027</partinfo> || I19|| DH5alpha || not detected<br />
|}<br />
<br />
'''Discussion''' These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in ''E. coli'' and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts.<br />
<br />
===<b>Modulation of plasmid copy number</b>===<br />
<br />
Signal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) beared on high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). <br />
The circuits we obtained and tested are summarized here:<br />
<br />
<br />
{| border='1' align='center'<br />
| '''BioBrick'''<br> '''Sender''' ||'''Description ''' || '''Sender Vector''' || '''<partinfo>BBa_F2620</partinfo><br> Receiver vector'''|| '''BioBrick composite part'''<br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan="2" align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC<br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| xxx|| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114<br />
|colspan='2' align='center'| <partinfo>pSB1A2</partinfo><br>HC <br />
|<partinfo>BBa_K300015</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300017</partinfo><br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300014</partinfo><br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300016</partinfo><br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
| colspan='2' align='center'| <partinfo>pSB4C5</partinfo><br>LC <br />
|<partinfo>BBa_K300012</partinfo><br />
|-<br />
| <partinfo>BBa_K300030</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300028</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300029</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300025</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300026</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|-<br />
| <partinfo>BBa_K300027</partinfo><br />
| [[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106<br />
|<partinfo>pSB4C5</partinfo><br>LC <br />
| <partinfo>pSB1A2</partinfo><br>HC <br />
| Parts are contained in two different vectors<br />
|}<br />
<br />
===<b>Results</b>===<br />
<br />
The following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:<br />
<br />
{| border='1' align='center'<br />
| '''Sender device'''<br />
| '''Sensor systems with GFP'''<br />
|'''Measurement Device'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|xxx<br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23114 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A2</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300023</partinfo><br>in <partinfo>pSB1A2</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300024</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300021</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300022</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|<partinfo>BBa_K300019</partinfo><br>in <partinfo>pSB4C5</partinfo> <br />
|-<br />
|<partinfo>BBa_K300030</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23118 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300028</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23110 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300029</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23116 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300026</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23105 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300025</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23101 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|-<br />
|<partinfo>BBa_K300027</partinfo> in <partinfo>pSB4C5</partinfo><br>[[Image:pv_SignalGeneratorDevice.png|150px]]<br>J23106 <br />
|<partinfo>BBa_T9002</partinfo> in <partinfo>pSB1A3</partinfo><br>[[Image:pv_T9002.png|300px]]<br />
|Sender and Receiver are contained<br> in two different plasmids, <br>cotransformed in the same cell<br />
|}<br />
<br />
Cultures of ''E. coli'' TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to [[Team:UNIPV-Pavia/Parts/Characterization#Microplate reader experiments for self-inducible promoters - Protocol #1|this protocol]] and all data collected were analyzed as explained in [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|this section]]<br />
<br />
{|<br />
|[[Image:pv_GrowthCurveSelf.png|400px|thumb|center|Growth curve of <partinfo>BBa_K300019</partinfo> (O.D.600)]]<br />
|[[Image:pv_FluoCurveSelf.png|400px|thumb|center|Fluorescence curve of <partinfo>BBa_K300019</partinfo> (G.F.P.)]]<br />
|-<br />
|[[Image:pv_FLUvsASB.png|400px|thumb|center|Fluorescence VS Optical density curve of <partinfo>BBa_K300019</partinfo>]]<br />
|[[Image:pv_Scell_Threshold.png|400px|thumb|center|Scell=(dGFP/dt)/O.D.600 and threshold]]<br />
|}<br />
<br />
Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving.<br />
<br />
For every self-inducible device, several parameters were evaluated:<br />
*O.D.start is the O.D.600 corresponding to the transcription initiation of the gene of interest, it was evaluated as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)|in this section]]<br />
*K_HSL is the HSL synthesys rate per cell, it was estimated with the algorithm described [[Team:UNIPV-Pavia/Parts/Characterization#Data Analysis to estimate the HSL synthesys rate per cell|here]]<br />
*Doubling time is the period of time required for a cell population to double. It was evaluated as described in [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|Doubling time evaluation section]]<br />
*Scell_ratio was evaluated as (Scell_max_Phi)/(Scell_max_J101). Phi is the self-inducible device of ineterst, J101 is the reference standard <partinfo>BBa_J23101</partinfo> contained in the same vector of the receiver device. Scell_max_phi was evaluated for times subsequent to the transcription initiation.<br />
<br />
Results are summarized in the following tables:<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br> [min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid</font> <br />
|[[Image:pv_BBa_K300017.png|300px]]<br />
| Constitutive<br />
| 3.11 10^-16 <br> ± <br> 2.23 10^-17<br />
| 31.15 <br> ± <br> 2.52<br />
| 0.95 <br> ± <br> 0.15<br />
| 0.027 <br> ± <br> 0.002<br />
| 1.52 10^-15 <br> ± <br> 9.76 10^-17<br />
| 61.28 <br> ± <br> 2.67<br />
| 1.68 <br> ± <br> 0.23<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 27.86 <br> ± <br> 1.14<br />
| <font color='red' size='+2'>X</font><br />
| 0.13 <br> ± <br> 0.023 **<br />
| 3.59 10^-16 <br> ± <br> 1.22 10^-16 **<br />
| 0.37 <br> ± <br> 0.13 **<br />
| 53.09 <br> ± <br> 1.18 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300015.png|300px]]<br />
| 0.33 <br> ± <br> *<br />
| 8.94 10^-17 <br> ± <br> *<br />
| 33.81 <br> ± <br> 3.03<br />
| 0.98 <br> ± <br> *<br />
| 0.38 <br> ± <br> 0.02<br />
| 3.78 10^-17 <br> ± <br> 4.65 10^-18<br />
| 57.20 <br> ± <br> 1.32<br />
| 0.07 <br> ± <br> 0.01<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300016.png|300px]]<br />
| 0.54 <br> ± <br> *<br />
| 7.53 10^-18 <br> ± <br> *<br />
| 39.55 <br> ± <br> 0.32<br />
| 0.21 <br> ± <br> *<br />
| 0.32 <br> ± <br> 0.02<br />
| 5.70 10^-17 <br> ± <br> 7.75 10^-18<br />
| 55.33 <br> ± <br> 5.19<br />
| 0.13 <br> ± <br> 0.02<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300012.png|300px]]<br />
| 0.50 <br> ± <br> 0.01<br />
| 1.16 10^-17 <br> ± <br> 6.41 10^-19<br />
| 36.66 <br> ± <br> 2.50<br />
| 0.58 <br> ± <br> 0.02<br />
| 0.30 <br> ± <br> 0.03<br />
| 6.53 10^-17 <br> ± <br> 1.43 10^-17<br />
| 50.92 <br> ± <br> 2.92<br />
| 0.21 <br> ± <br> 0.06<br />
|}<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL''' <br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time''' <br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300017.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 0.24 <br> ± <br> 0.0004 **<br />
| not computed<br />
| 62.41 <br> ± <br> 1.56 **<br />
| not computed<br />
|-<br />
|<font color='red'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300014.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='red'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300015.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300016.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|- <br />
|<font color='red'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300012.png|300px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|} <br />
<br />
'''Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>'''<br />
<br />
{| border='1'<br />
|rowspan='2' align='center'|'''Self-inducible device''' <br><font color='#50C878'>working</font><br><font color='red'>not working</font><br />
|rowspan='2' align='center' colspan='2'|'''Description''' <br />
|colspan='4' style="background: yellow" align='center' |LB<br />
|colspan='4' style="background: cyan" align="center" |M9<br />
|-<br />
|align='center'| '''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'| '''Scell ratio'''<br />
|align='center'|'''O.D.start''' <br />
|align='center'| '''K_HSL'''<br> [nmol/min]<br />
|align='center'| '''Doubling time'''<br>[min]<br />
|align='center'|'''Scell ratio'''<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300030.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| Constitutive<br />
| 2.92 10^-16 <br> ± <br> 8.16 10^-18<br />
| 33.20 <br> ± <br> 1.46<br />
| 1.20 <br> ± <br> 0.20<br />
| 0.05 <br> ± <br> 0.005<br />
| 8.06 10^-16 <br> ± <br> 1.28 10^-16<br />
| 58.27 <br> ± <br> 5.66<br />
| 0.91 <br> ± <br> 0.15<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300028.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| 0.32 <br> ± <br> 0.04 **<br />
| 8.31 10^-17 <br> ± <br> 3.92 10^-17 **<br />
| 33.24 <br> ± <br> 1.27<br />
| 0.61 <br> ± <br> 0.04 **<br />
| 0.15 <br> ± <br> 0.007<br />
| 1.72 10^-16 <br> ± <br> 6.65 10^-18<br />
| 65.57 <br> ± <br> 5.99<br />
| 0.26 <br> ± <br> 0.03<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300029</partinfo><br> (wiki name: I16)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300029.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| 0.31 <br> ± <br> *<br />
| 1.17 10^-16 <br> ± <br> *<br />
| 35.46 <br> ± <br> 2.85<br />
| 0.57 <br> ± <br> *<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300025.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| Constitutive<br />
| 2.85 10^-16 <br> ± <br> 1.90 10^-17<br />
| 33.27 <br> ± <br> 2.85<br />
| 1.09 <br> ± <br> 0.28<br />
| 0.1 <br> ± <br> 0.01<br />
| 3.81 10^-16 <br> ± <br> 4.68 10^-17<br />
| 59.02 <br> ± <br> 8.28<br />
| 0.45 <br> ± <br> 0.08<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300026.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 34.63 <br> ± <br> 1.31<br />
| <font color='red' size='+2'>X</font><br />
| 0.53 <br> ± <br> 0.03 **<br />
| 1.78 10^-17 <br> ± <br> 1.36 10^-18 **<br />
| 61.68 <br> ± <br> 7.08<br />
| 0.03 <br> ± <br> 0.002 **<br />
|-<br />
|<font color='#50C878'><partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid</font><br />
|[[Image:pv_BBa_K300027.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| 33.80 <br> ± <br> 1.78<br />
| <font color='red' size='+2'>X</font><br />
| 0.33 <br> ± <br> 0.05<br />
| 5.46 10^-17 <br> ± <br> 7.86 10^-18<br />
| 52.84 <br> ± <br> 4.29<br />
| 0.06 <br> ± <br> 0.002<br />
|}<br />
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LEGEND OF TABLES:<br />
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''Constitutive'': the induction point, in term of O.D.600, is under the minimum detectable value calculated by the aglorithm. This minimum value was estimated by running the algorithm on data acquired from a culture that constitutively produces GFP. For this reason, the devices labelled as ''constitutive'' can be considered as constitutive GFP producers.<br />
<br />
<nowiki>*</nowiki>: in two of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were not computed for these cultures.<br />
<br />
<nowiki>**</nowiki>: in one of three experiments the self-induction failed, thus having a non-induced culture for all the cell densities. The standard errors were computed computed on two independent experiments.<br />
<br />
<partinfo>BBa_K300016</partinfo> is labelled with <nowiki>*</nowiki>, but probably induction failed in two of the three experiments because the culture didn't reach the ODstart point (the experiment was stopped before the culture reached the O.D.600 critical value).<br />
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<br />
'''Discussion''': a self-inducible device (<partinfo>BBa_K300010</partinfo>) was realized and characterized in many different experimental conditions:<br />
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation)<br />
*varying the copy number of vectors bearing both Sender and Receiver circuits<br />
*varying the growth medium (LB or M9)<br />
<br />
A library of self-inducible promoters, able to start the production of the heterologous protein at a defined culture density, was realized. A graphical summary is reported in Figures:<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|-<br />
|[[Image:pv_SwitchPointM9.png|700px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
A model-based approach was proposed to estimate many interesting parameters, such as the HSL synthesis rate per cell. <br />
<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div></div>Susanna