http://2010.igem.org/wiki/index.php?title=Special:Contributions/Matteo&feed=atom&limit=50&target=Matteo&year=&month=2010.igem.org - User contributions [en]2024-03-29T14:02:45ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistryTeam:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry2010-10-28T03:42:09Z<p>Matteo: </p>
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<html><p align="center"><font size="4"><b>EXISTING PARTS FROM THE REGISTRY<br>(all these reported informations are shared in the Registry of Standard 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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=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 />
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 />
<br />
The measurement system of this part (<partinfo>BBa_T9002</partinfo>) was successfully used as a biosensor to estimate the concentration of HSL present in the growth media of cultures expressing ''luxI'' (<partinfo>BBa_C0061</partinfo>).<br />
<br />
Additional information and details are available in [http://partsregistry.org/Part:BBa_K300009:Experience BBa_K300009 Experience page].<br />
<br />
<br />
{| align='center'<br />
|[[Image:pv_HCT9002sensor.png|500px|thumb|center|<partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced by autoinducer generators in high copy vector.]]<br />
|}<br />
<br />
<br />
{| align='center'<br />
|[[Image:pv_LCT9002sensor.png|500px|thumb|center|<partinfo>BBa_T9002</partinfo> calibration curve for detection of [HSL] produced by the autoinducer generators in low copy vector.]]<br />
|}<br />
<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>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewPartsTeam:UNIPV-Pavia/Parts/Characterization/NewParts2010-10-28T03:14:22Z<p>Matteo: /* New Parts: 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" 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_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, BBa_K300086 and BBa_K300084 - Phasin and Intein-based tags for protein purification|BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095, BBa_K300086 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_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 />
Notation:<br />
*Sender: Pcon-RBS-luxI-TT<br />
*Receiver: pTetR-RBS-luxR-TT- lux pR<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 />
<partinfo>BBa_J23101</partinfo> and <partinfo>BBa_J23106</partinfo> 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 />
Sender and receiver devices 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>. <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 other examples for the threshold evaluation, you can see:<br />
*<partinfo>BBa_K300012</partinfo><br />
*<partinfo>BBa_K300016</partinfo><br />
*<partinfo>BBa_K300028</partinfo><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 members 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>, <partinfo>BBa_K300086</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_K300086</partinfo><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_K300086</partinfo><br />
*<partinfo>BBa_K300088</partinfo><br />
*<partinfo>BBa_K300090</partinfo><br />
*<partinfo>BBa_K300099</partinfo><br />
*<partinfo>BBa_K173000</partinfo> (positive control, J23100 constitutive promoter expressing GFP)<br />
*<partinfo>BBa_B0031</partinfo> (negative control, a non-fluorescent culture)<br />
Cultures were grown 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 />
The optical density (O.D.) of each 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 />
Then we performed a 21-hour experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200; cultures were shaken for 15 seconds every five minutes. Acquired data were blanked by subtracting the media absorbance (for absorbance measurements) and the <partinfo>BBa_B0031</partinfo> fluorescence (for fluorescence measurements). Then, the relative GFP synthesis rate per cell was evaluated by computing (1/O.D.600)*dGFP/dt, where O.D.600 is the blanked absorbance of the culture of interest and GFP is its blanked fluorescence.<br />
Each value shown below is the mean of three measurements in exponential phase and error bars represent the 95% confidence interval of the mean.<br />
<br />
====Results====<br />
<div align="center"><br />
<table border="0"><br />
<tr><br />
<td>[[Image:UNIPV10_pTET_c_ASB.png|thumb|300px|Raw growth curve]]</td><br />
<td>[[Image:UNIPV10_pTET_c_GFP.png|thumb|300px|Raw GFP curve]]</td><br />
</tr><br />
</table><br />
<table><br />
<tr><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 />
</table><br />
<br />
<table border="1"><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_K300086</partinfo></td><td>73.6685 ± 1.6245</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 />
</div><br />
<br />
====Discussion====<br />
All the cultures showed a similar growth curve; doubling time was computed as described [https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation here] in order to obtain information about the metabolic burden due to the synthesis of the studied fusion proteins. It is possible to see that all doubling times are comparable; it is possible to assert that the expression of these BioBrick parts doesn't cause abnormal stress to the cells.<br />
<br />
From GFP curve it is possible to appreciate that in <partinfo>BBa_K300086</partinfo>, <partinfo>BBa_K300088</partinfo>, <partinfo>BBa_K300090</partinfo>, <partinfo>BBa_K300099</partinfo> GFP accumulation is very similar and it is significantly different from the one of the negative control <partinfo>BBa_B0031</partinfo>. These results show that the green fluorescent protein assembled downstream of the genetic circuit is correctly folded.<br />
<br />
The mean protein synthesis rate was also computed over the exponential growth phase, showing again an appreciable GFP production rate that is about half of the positive control GFP.<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 />
Cultures were grown 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 />
The optical density (O.D.) of each 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 />
Then we performed a 21-hour experiment with measurements of absorbance and green fluorescence every five minutes using TECAN Infinite F200; cultures were shaken for 15 seconds every five minutes. <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> constructs were induced with 100nM of HSL directly in the 96-well microplate. Acquired data were blanked by subtracting the media absorbance (for absorbance measurements) and the <partinfo>BBa_B0031</partinfo> fluorescence (for fluorescence measurements). Then, the relative GFP synthesis rate per cell was evaluated by computing (1/O.D.600)*dGFP/dt, where O.D.600 is the blanked absorbance of the culture of interest and GFP is its blanked fluorescence.<br />
Each value shown below is the mean of three measurements in exponential phase and error bars represent the 95% confidence interval of the mean.<br />
<br />
====Results====<br />
<div align="center"><br />
<table><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><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"><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 />
</div><br />
<br />
====Discussion====<br />
All the cultures showed a similar growth curve; doubling time was computed as described [https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation here] in order to obtain information about the burden due to the synthesis of such fusion proteins. It is possible to see that all doubling times are very similar except for induced cultures. In this case doubling time is much higher than both positive control and non-induced cultures; for this reason it is possible to assert that induction gives a high metabolic burden.<br />
<br />
From GFP curve and mean protein synthesis rate it is possible to appreciate that induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> GFP accumulation profiles are comparable and they significantly differ from the GFP raw time series of the negative control <partinfo>BBa_B0031</partinfo>. On the other hand not induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> show a profile that is very similar to the negative control. These results show that the green fluorescent protein assembled downstream of the construct is correctly folded and that the inducible system works as expected.<br />
<br />
Not induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> show a low GFP synthesis rate maybe due to 3OC6HSL inducible <br />
<br />
</td><br />
</tr></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewPartsTeam:UNIPV-Pavia/Parts/Characterization/NewParts2010-10-28T02:11:34Z<p>Matteo: /* 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, BBa_K300086 and BBa_K300084 - Phasin and Intein-based tags for protein purification|#BBa_K300093, BBa_K300094, BBa_K300097, BBa_K300095, BBa_K300086 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_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 />
Notation:<br />
*Sender: Pcon-RBS-luxI-TT<br />
*Receiver: pTetR-RBS-luxR-TT- lux pR<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 />
<partinfo>BBa_J23101</partinfo> and <partinfo>BBa_J23106</partinfo> 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 />
Sender and receiver devices 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>. <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 other examples for the threshold evaluation, you can see:<br />
*<partinfo>BBa_K300012</partinfo><br />
*<partinfo>BBa_K300016</partinfo><br />
*<partinfo>BBa_K300028</partinfo><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 members 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>, <partinfo>BBa_K300086</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_K300086</partinfo><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_K300086</partinfo><br />
*<partinfo>BBa_K300088</partinfo><br />
*<partinfo>BBa_K300090</partinfo><br />
*<partinfo>BBa_K300099</partinfo><br />
*<partinfo>BBa_K173000</partinfo> (positive control, J23100 constitutive promoter expressing GFP)<br />
*<partinfo>BBa_B0031</partinfo> (negative control, a non-fluorescent culture)<br />
Cultures were grown 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 />
The optical density (O.D.) of each 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 />
Then we performed a 21-hour experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200; cultures were shaken for 15 seconds every five minutes. Acquired data were blanked by subtracting the media absorbance (for absorbance measurements) and the <partinfo>BBa_B0031</partinfo> fluorescence (for fluorescence measurements). Then, the relative GFP synthesis rate per cell was evaluated by computing (1/O.D.600)*dGFP/dt, where O.D.600 is the blanked absorbance of the culture of interest and GFP is its blanked fluorescence.<br />
Each value shown below is the mean of three measurements in exponential phase and error bars represent the 95% confidence interval of the mean.<br />
<br />
====Results====<br />
<div align="center"><br />
<table border="0"><br />
<tr><br />
<td>[[Image:UNIPV10_pTET_c_ASB.png|thumb|300px|Raw growth curve]]</td><br />
<td>[[Image:UNIPV10_pTET_c_GFP.png|thumb|300px|Raw GFP curve]]</td><br />
</tr><br />
</table><br />
<table><br />
<tr><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 />
</table><br />
<br />
<table border="1"><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_K300086</partinfo></td><td>73.6685 ± 1.6245</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 />
</div><br />
<br />
====Discussion====<br />
All the cultures showed a similar growth curve; doubling time was computed as described [https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation here] in order to obtain information about the metabolic burden due to the synthesis of the studied fusion proteins. It is possible to see that all doubling times are comparable; it is possible to assert that the expression of these BioBrick parts doesn't cause abnormal stress to the cells.<br />
<br />
From GFP curve it is possible to appreciate that in <partinfo>BBa_K300086</partinfo>, <partinfo>BBa_K300088</partinfo>, <partinfo>BBa_K300090</partinfo>, <partinfo>BBa_K300099</partinfo> GFP accumulation is very similar and it is significantly different from the one of the negative control <partinfo>BBa_B0031</partinfo>. These results show that the green fluorescent protein assembled downstream of the genetic circuit is correctly folded.<br />
<br />
The mean protein synthesis rate was also computed over the exponential growth phase, showing again an appreciable GFP production rate that is about half of the positive control GFP.<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 />
Cultures were grown 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 />
The optical density (O.D.) of each 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 />
Then we performed a 21-hour experiment with measurements of absorbance and green fluorescence every five minutes using TECAN Infinite F200; cultures were shaken for 15 seconds every five minutes. <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> constructs were induced with 100nM of HSL directly in the 96-well microplate. Acquired data were blanked by subtracting the media absorbance (for absorbance measurements) and the <partinfo>BBa_B0031</partinfo> fluorescence (for fluorescence measurements). Then, the relative GFP synthesis rate per cell was evaluated by computing (1/O.D.600)*dGFP/dt, where O.D.600 is the blanked absorbance of the culture of interest and GFP is its blanked fluorescence.<br />
Each value shown below is the mean of three measurements in exponential phase and error bars represent the 95% confidence interval of the mean.<br />
<br />
====Results====<br />
<div align="center"><br />
<table><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><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"><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 />
</div><br />
<br />
====Discussion====<br />
All the cultures showed a similar growth curve; doubling time was computed as described [https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation here] in order to obtain information about the burden due to the synthesis of such fusion proteins. It is possible to see that all doubling times are very similar except for induced cultures. In this case doubling time is much higher than both positive control and non-induced cultures; for this reason it is possible to assert that induction gives a high metabolic burden.<br />
<br />
From GFP curve and mean protein synthesis rate it is possible to appreciate that induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> GFP accumulation profiles are comparable and they significantly differ from the GFP raw time series of the negative control <partinfo>BBa_B0031</partinfo>. On the other hand not induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> show a profile that is very similar to the negative control. These results show that the green fluorescent protein assembled downstream of the construct is correctly folded and that the inducible system works as expected.<br />
<br />
Not induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> show a low GFP synthesis rate maybe due to 3OC6HSL inducible <br />
<br />
</td><br />
</tr></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistryTeam:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry2010-10-27T23:59:24Z<p>Matteo: </p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3">{{UNIPV-Pavia/header}}</td><br />
</tr><br />
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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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<table class="cont" border="2" width="100%" align="center"><br />
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[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 />
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[https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br />
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<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_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>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistryTeam:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry2010-10-27T23:53:10Z<p>Matteo: </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 />
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<!-- 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 the characterization results reported below are shared 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_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>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistryTeam:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry2010-10-27T23:23:34Z<p>Matteo: </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_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>Matteohttp://2010.igem.org/File:Pv_MinimumInduction.pngFile:Pv MinimumInduction.png2010-10-27T08:45:54Z<p>Matteo: </p>
<hr />
<div></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Material_Methods/Measurements/TecanTeam:UNIPV-Pavia/Material Methods/Measurements/Tecan2010-10-26T11:10:40Z<p>Matteo: </p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="3" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><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>TECAN TEST</b></font></p></html><hr><br><br />
<table class=cont border="0" width="30%" align="center" valign="top"><br />
<tr><th align="center" width="12%"><b>DATE</b></th><th align="center"><b>DESCRIPTION</b></th></tr><br />
<br />
<br />
<br />
<tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/August/settimana3#August, 17th|August, 17th]] </td><td align="left"><br />
Test on self-inducible promoters (LB) [[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test17agosto|See more...]]<br />
</td></tr><br />
<tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/August/settimana3#August, 18th|August, 18th]] </td><td align="left"><br />
Test on self-inducible promoters (LB) [[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test18agosto|See more...]]<br />
</td></tr><br />
<tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/August/settimana3#August, 20th|August, 20th]] </td><td align="left"><br />
Test on self-inducible promoters (LB) [[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test20agosto|See more...]]<br />
</td></tr><br />
<tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/August/settimana4#August, 27th|August, 27th]] </td><td align="left"><br />
HC promoters (LB) [[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test27agosto|See more...]]<br />
</td></tr><br />
<tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/September/settimana1#September, 1st|September, 1st]] </td><td align="left"><br />
Test on self-inducible promoters (M9) [[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test1settembre|See more...]]<br />
</td></tr><br />
<tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/September/settimana1#September, 2nd|September, 2nd]] </td><td align="left"><br />
HC and LC promoters test (M9) [[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test2settembre|See more...]]<br />
</td></tr><br />
<tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/September/settimana2#September, 8th|September, 8th]] <br />
</td><td align="left"><br />
3OC6-HSL generators test [[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test8settembre|See more...]]<br />
</td></tr><br />
<tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/September/settimana2#September, 10th|September, 10th]] <br />
</td><td align="left"><br />
3OC6-HSL generators test [[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test10settembre_bis|See more...]]<br />
</td></tr><br />
<tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/September/settimana3#September, 14th|September, 14th]] <br />
</td><td align="left"><br />
LC promoters (LB) [[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test14settembre|See more...]]<br />
</td></tr><br />
<tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/September/settimana3#September, 16th|September, 16th]]<br />
</td><td align="left"><br />
Test on self-inducible promoters with a different protocol (LB) [[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test16settembre|See more...]]<br />
</td></tr><br />
<br />
<tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/September/settimana5#September, 27th|September, 27th]]<br />
</td><td align="left"><br />
I47, I48, I49 GFP synthesis test. <partinfo>BBa_K173002</partinfo> as positive control, <partinfo>BBa_B0031</partinfo> as negative control.<br />
<br/><br />
[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test27settembre|See more...]]<br />
</td></tr><br />
<tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/September/settimana5#September, 28th|September, 28th]]<br />
</td><td align="left"><br />
I63 (induced and not induced), I64 (induced and not induced), I65 GFP synthesis test. <partinfo>BBa_K173002</partinfo> as positive control, <partinfo>BBa_B0031</partinfo> as negative control.<br />
<br/><br />
[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test28settembre|See more...]]<br />
</td></tr><br />
<tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/September/settimana5#September, 30th|September, 30th]]<br />
</td><td align="left"><br />
Test on self-inducible promoters (M9) [[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test30settembre|See more...]]<br />
</td></tr><br />
<tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/September/settimana1#October, 1st|October, 1st]]<br />
</td><td align="left"><br />
Test on self-inducible promoters (M9) [[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test1ottobre|See more...]]<br />
</td></tr><br />
</table><br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
</table></div>Matteohttp://2010.igem.org/File:Pv_P1P4singlecloneintegrant.pngFile:Pv P1P4singlecloneintegrant.png2010-10-26T08:47:15Z<p>Matteo: </p>
<hr />
<div></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Project/resultsTeam:UNIPV-Pavia/Project/results2010-10-26T08:39:20Z<p>Matteo: /* Chloramphenicol resistance marker excision */</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="6" align="center" valign="top"><b>Implementation and Results</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><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UNIPV_Pavia_Icona_PromotoriAuto.GIF" width="55px" height="70px"/><br />
</html><br />
<br><br />
[[#Self-inducible prometers|Self-inducible promoters]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/UNIPV_Pavia_Icona_intColi.GIF" width="55px" height="70px"/><br />
</html><br />
<br><br />
[[#Integrative standard vector for E. coli|Integrative standard vector for E. coli]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/0/03/UNIPV_Pavia_Icona_IntYeast.GIF" width="55px" height="70px"/><br />
</html><br />
<br><br />
[[#Integrative standard vector for yeast|Integrative standard vector for yeast]]<br />
</td><br />
<td align="center" valign = "center" style="padding:20px" width="25%"><br />
<html><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 />
[[#Self-cleaving affinity tags to easily purify proteins|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 Data Analysis RPU; <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 />
'''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> sensor was contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10.<br />
<br />
'''Results''' The amount of 3OC6-HSL produced after 6 hours growth by ''E. coli'' DH5alpha bearing the parts contained in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Figure 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 />
{| <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 Figure 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 />
{| <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 generator to produce 3OC6-HSL signal molecule. Data are partially uncompleted 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, even if the correspondence is not exact, probably due to the fact that very strong promoters (such as <partinfo>BBa_J23101</partinfo>) give to the cells a high metabolic burden, thus resulting in lower production yields.<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 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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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 />
Doubling times were estimated as explained [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|here]]<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, as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)| in this section]]. Results are summarized in the following tables:<br />
<br />
<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1'<br />
|'''Self-inducible device''' || '''Description''' || '''O.D.start LB''' || '''K_HSL LB'''|| '''Doubling time LB'''|| '''O.D.start M9''' || '''K_HSL M9'''|| '''Doubling time M9'''<br />
|-<br />
|<partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid <br />
|[[Image:pv_BBa_K300017.png|400px]]<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid<br />
|[[Image:pv_BBa_K300014.png|400px]]<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid<br />
|[[Image:pv_BBa_K300015.png|400px]]<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid<br />
|[[Image:pv_BBa_K300016.png|400px]]<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid<br />
|[[Image:pv_BBa_K300012.png|400px]]<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|}<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
{| border='1'<br />
|'''Self-inducible device''' || '''Description''' || '''O.D.start LB''' || '''K_HSL LB'''|| '''Doubling time LB'''|| '''O.D.start M9''' || '''K_HSL M9'''|| '''Doubling time M9'''<br />
|-<br />
|<partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid <br />
|[[Image:pv_BBa_K300017.png|400px]]<br />
| <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 />
|-<br />
|<del style='color:#C2DFFF'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</del><br />
|[[Image:pv_BBa_K300014.png|400px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <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 />
|<del style='color:#C2DFFF'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</del><br />
|[[Image:pv_BBa_K300015.png|400px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <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 />
|<del style='color:#C2DFFF'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</del><br />
|[[Image:pv_BBa_K300016.png|400px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <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 />
|<del style='color:#C2DFFF'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</del><br />
|[[Image:pv_BBa_K300012.png|400px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <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 />
|'''Self-inducible device''' <br />
| '''Sender Description'''<br />
| '''Receiver Description''' <br />
| '''O.D.start LB''' <br />
| '''K_HSL LB'''<br />
| '''Doubling time LB'''<br />
| '''O.D.start M9''' <br />
| '''K_HSL M9'''<br />
| '''Doubling time M9'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300030.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300028.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300029</partinfo><br> (wiki name: I16)<br> in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300029.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300025.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300026.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300027.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|}<br />
<br />
<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|400px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|[[Image:pv_SwitchPointM9.png|400px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/UNIPV_Pavia_Icona_intColi.GIF" width="55px" height="70px"/><br />
</html><br />
<br />
=Integrative standard 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). 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). DH5alpha (<partinfo>BBa_V1001</partinfo>) strain was purchased from Invitrogen.<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 as:<br />
<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 />
<br />
'''Pir strains validation:''' in order to test the capability of BW25141 (pir+) and BW23474 (pir-116) to propagate this conditional replication origin, they were made competent, as well as three control strains: MC1061, MG1655 and DH5alpha. 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> (a R6K plasmid with Cm resistance).<br />
<br />
Self-ligated <partinfo>BBa_K300008</partinfo> was prepared by digesting <partinfo>pSB1A2</partinfo>-<partinfo>BBa_K300008</partinfo> (yielded by BioBrick Standard Assembly) with XbaI-SpeI. The insert was isolated and purified from a 1% agarose gel. Then, it was self-ligated to generate a Cm-resistant R6K plasmid. The colonies were counted in each plate and the transformation efficiency was estimated as described before.<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 />
'''BBa_K300000 construction:''' This section describes how this vector backbone was assembled using BioBrick parts.<br />
#'''First step - <partinfo>BBa_K300982</partinfo> intermediate part construction:'''<br />
##<partinfo>BBa_K300983</partinfo>, provided by Mr Gene DNA synthesis service (www.mrgene.com), was excised from its original shipping vector (pMK-RQ) through digestion with AvrII restriction enzyme (Roche). It was then isolated through a 1% agarose gel electrophoresis and gel-extracted (Macherey-Nagel NucleoSpin Extract II).<br />
##<partinfo>BBa_K300008</partinfo> was assembled via BioBrick Standard Assembly from available parts and was excised from its vector (<partinfo>pSB1A2</partinfo>) through digestion with XbaI-SpeI (Roche). It was then isolated using the same procedure described above and it was dephosphorylated by using Antarctic Phosphatase (NEB).<br />
##Digested <partinfo>BBa_K300983</partinfo> and <partinfo>BBa_K300008</partinfo>, all having compatible sticky ends, were ligated with T4 Ligase (Roche) and transformed into competent BW23474 E. coli strain (<partinfo>BBa_K300985</partinfo>) in order to allow the ligated plasmid propagation at high copy number. This strain was necessary because the replication origin of the resulting plasmid was the conditional R6K origin (<partinfo>BBa_J61001</partinfo>).<br />
##Positive transformants, grown on Chloramphenicol (34 ug/ml) plates, were identified by restriction mapping with EcoRI-HindIII (Roche). The yielded plasmid was <partinfo>BBa_K300982</partinfo>.<br />
#'''Second step - <partinfo>BBa_K300000</partinfo> final vector construction:'''<br />
##<partinfo>BBa_K300982</partinfo> DNA was miniprepped, digested with EcoRI-PstI (Roche), run on agarose gel and gel-extracted as described above, in order to cut out and eliminate the RBS (<partinfo>BBa_B0033</partinfo>) between EcoRI and PstI.<br />
##<partinfo>BBa_I52002</partinfo> DNA was excised from <partinfo>pSB4C5</partinfo> by EcoRI-PstI (Roche) digestion and isolated by gel run and gel extraction as described above.<br />
##Digested <partinfo>BBa_K300982</partinfo> and <partinfo>BBa_I52002</partinfo> were ligated and transformed into competent DB3.1 E. coli strain (<partinfo>BBa_V1005</partinfo>), selected with Chloramphenicol at 34 ug/ml.<br />
##Positive transformants were screened by restriction mapping with EcoRI-HindIII (Roche) and sequencing with VF2 (<partinfo>BBa_G00100</partinfo>) and VR (<partinfo>BBa_G00101</partinfo>) standard primers. The yielded plasmid was <partinfo>BBa_K300000</partinfo>.<br />
<br />
All the DNA manipulations were performed according to manufacturer's protocols.<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 />
===Validation of pir strains to propagate the R6K replication origin===<br />
The BW25141 (pir+) and BW23474 (pir-116) ''E. coli'' strains were chosen to propagate the vectors with the R6K replication origin at medium copy (~15 molecules per cell) and high copy (~250) respectively.<br />
The results about their capability to propagate R6K plasmids are shown here:<br />
<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 />
|BW25141 (<partinfo>BBa_K300084</partinfo>, pir+)<br />
|0<br />
|10^5<br />
|10^5<br />
|-<br />
|BW23474 (<partinfo>BBa_K300085</partinfo>, pir-116)<br />
|0<br />
|10^6<br />
|10^6<br />
|-<br />
|DH5alpha (<partinfo>BBa_V1001</partinfo>, neg control)<br />
|0<br />
|10^8<br />
|0<br />
|-<br />
|MC1061 (<partinfo>BBa_K300078</partinfo>, neg control)<br />
|0<br />
|10^6<br />
|0<br />
|-<br />
|MG1655 (<partinfo>BBa_V1000</partinfo>, neg control)<br />
|0<br />
|10^5<br />
|six small colonies from 2 ng of DNA<br />
|}<br />
<br />
<br />
These results show that the R6K conditional 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. The exception was MG1655, for which six small unwanted colonies appeared on the selective plate. Plasmid DNA was purified/HindIII-digested/gel-run for at least one colony for BW25141-<partinfo>BBa_K300008</partinfo> and BW23474-<partinfo>BBa_K300008</partinfo>, while all the six colonies were analyzed for MG1655-<partinfo>BBa_K300008</partinfo> plate. The electrophoresis showed the expected length of the transformed DNA for all the clones except for the MG1655 six colonies, for which a smear was present in the lane (data not shown). Probably, Cm-resistant contaminants were present in the MG1655 culture during the preparation of competent cells.<br />
<br />
For this reason, competent cells were prepared again for MG1655 and the transformation procedure was repeated for this strain, yielding no colonies in the <partinfo>BBa_K300008</partinfo> plate as expected.<br />
<br />
Miniprep of the BW25141 and BW23474 strains transformed with <partinfo>BBa_K300008</partinfo> yielded a DNA concentration of ~20 ng/ul (qualitatively comparable with medium copy number plasmids) and ~90-100 ng/ul (qualitatively comparable with high copy number plasmids).<br />
The results shown in the table above also 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 />
So, the BW25141 and BW23474 strains can be successfully used to propagate the integrative vector after the excision of the pUC19-derived high copy replication origin, present in the default insert <partinfo>BBa_I52002</partinfo>.<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 />
<div align="right"><small>[[#indice|^top]]</small></div><br />
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<br />
=Integrative standard vector for yeast=<br />
<br />
==Materials and Methods==<br />
<br />
'''Strain:''' the ''S. cerevisiae'' S288C strain (<partinfo>BBa_K300979</partinfo>, genotype: MATα ρ° trp1-0) was purchased from Open Biosystems.<br />
<br />
'''Construction of BBa_K300001:'''<br />
<br />
#<partinfo>BBa_K300980</partinfo>, provided by Mr Gene DNA synthesis service (www.mrgene.com), was excised from its original shipping vector (pMA) through digestion with MfeI (Fermentas) and NsiI (Fermentas) restriction enzymes. It was then isolated through a 1% agarose gel electrophoresis and gel-extracted (Macherey-Nagel NucleoSpin Extract II).<br />
#<partinfo>BBa_I763007</partinfo>, available in the Registry, was digested with EcoRI-PstI (Roche), ran on agarose gel and its <partinfo>pSB1A2</partinfo> vector was gel-extracted as described above.<br />
#Digested <partinfo>BBa_K300980</partinfo> and <partinfo>pSB1A2</partinfo> have compatible ends (EcoRI-MfeI and PstI-NsiI). They were ligated with T4 Ligase (Roche) and transformed into competent TOP10 E. coli strain (<partinfo>BBa_V1009</partinfo>) which were then plated on Ampicillin (100 ug/ml) plates.<br />
#Positive transformants were identified by colony PCR with VF2 (<partinfo>BBa_G00100</partinfo>) and VR (<partinfo>BBa_G00101</partinfo>) standard primers and by restriction mapping with EcoRI (Roche) or NsiI (Fermentas). The yielded plasmid had <partinfo>BBa_B0033</partinfo> flanked by EcoRI and SpeI.<br />
#The yielded plasmid was then digested with EcoRI-SpeI (Roche) and <partinfo>BBa_K300007</partinfo> (digested with EcoRI-SpeI as well) was assembled in the vector, thus yielding <partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300007</partinfo> (i.e. there is the <partinfo>BBa_K300007</partinfo> part in the cloning site).<br />
<br />
<br />
'''Yeast transformation:'''<br />
<br />
#S288C strain 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 />
==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 />
|6500<br />
|6.5*10^3<br />
|-<br />
|<partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300007</partinfo><br />
|1<br />
|1700<br />
|1.7*10^3<br />
|-<br />
|no DNA<br />
|0<br />
|0<br />
|0<br />
|}<br />
<br />
The colony count was quite difficult (see pictures), but it is useful to have an estimation of the integration efficiency.<br />
<br />
{|align=center<br />
|[[Image:pv_yeastnodna.jpg|thumb|200px|center|S288C transformed with no DNA (negative control).]]<br />
|[[Image:pv_yeast06.jpg|thumb|200px|center|S288C transformed with linearized <partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300006</partinfo>.]]<br />
|[[Image:pv_yeast07.jpg|thumb|200px|center|S288C transformed with linearized <partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300007</partinfo>.]]<br />
|}<br />
<br />
==Discussion==<br />
<br />
The obtained 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 />
Although this integrative vector has already given promising integration results, a lot of work still remains to do:<br />
* the correct phenotype of the S288C bearing these parts has to be validated (by mOrange fluorescence measurement for the <partinfo>BBa_K300007</partinfo> part);<br />
* the actual integration position has to be confirmed by PCR with ad hoc-designed primers;<br />
* the KanMX selection marker excision has to be validated.<br />
<br />
<br />
The compatibility with the BioBrick standard (RFC10, RFC12 and RFC23) enables the engineering the chromosomes of ''S. cerevisiae'' by integrating the desired biological functions following a standard procedure. Moreover, the modularity of the designed integrative vector enables the modification of the integration target site, in fact the default Homologous Regions can only target the Gal genomic system and users may need to target different DNA sequences.<br />
<br />
As reported for the integrative vector for ''E. coli'', the main information about the usage of this integrative vector for yeast is shared in the Registry. We hope to have positively contributed to the diffusion of standard biological parts for yeast, a very interesting chassis for synthetic biology for which only a limited number of parts are available in the Registry and only a few of them has been exaustively characterized.<br />
<br />
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<br />
=Self-cleaving affinity tags to easily purify proteins=<br />
{{UNIPV-Pavia/Project/results/Self-cleaving_affinity_tags_to_easily_purify_proteins}}<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
</td></tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Project/resultsTeam:UNIPV-Pavia/Project/results2010-10-26T08:36:46Z<p>Matteo: /* Chloramphenicol resistance marker excision */</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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<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="6" align="center" valign="top"><b>Implementation and Results</b></font><br />
</p><br />
<br><br />
<hr><br />
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<br><br />
[[#Self-inducible prometers|Self-inducible promoters]]<br />
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[[#Integrative standard vector for E. coli|Integrative standard vector for E. coli]]<br />
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[[#Integrative standard vector for yeast|Integrative standard vector for yeast]]<br />
</td><br />
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<br><br />
[[#Self-cleaving affinity tags to easily purify proteins|Purification of proteins]]<br />
</td><br />
</tr><br />
</table><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 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 Data Analysis RPU; <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 />
'''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> sensor was contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10.<br />
<br />
'''Results''' The amount of 3OC6-HSL produced after 6 hours growth by ''E. coli'' DH5alpha bearing the parts contained in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Figure 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 />
{| <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 Figure 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 />
{| <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 generator to produce 3OC6-HSL signal molecule. Data are partially uncompleted 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, even if the correspondence is not exact, probably due to the fact that very strong promoters (such as <partinfo>BBa_J23101</partinfo>) give to the cells a high metabolic burden, thus resulting in lower production yields.<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 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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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 />
Doubling times were estimated as explained [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|here]]<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, as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)| in this section]]. Results are summarized in the following tables:<br />
<br />
<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1'<br />
|'''Self-inducible device''' || '''Description''' || '''O.D.start LB''' || '''K_HSL LB'''|| '''Doubling time LB'''|| '''O.D.start M9''' || '''K_HSL M9'''|| '''Doubling time M9'''<br />
|-<br />
|<partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid <br />
|[[Image:pv_BBa_K300017.png|400px]]<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid<br />
|[[Image:pv_BBa_K300014.png|400px]]<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid<br />
|[[Image:pv_BBa_K300015.png|400px]]<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid<br />
|[[Image:pv_BBa_K300016.png|400px]]<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid<br />
|[[Image:pv_BBa_K300012.png|400px]]<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|}<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
{| border='1'<br />
|'''Self-inducible device''' || '''Description''' || '''O.D.start LB''' || '''K_HSL LB'''|| '''Doubling time LB'''|| '''O.D.start M9''' || '''K_HSL M9'''|| '''Doubling time M9'''<br />
|-<br />
|<partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid <br />
|[[Image:pv_BBa_K300017.png|400px]]<br />
| <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 />
|-<br />
|<del style='color:#C2DFFF'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</del><br />
|[[Image:pv_BBa_K300014.png|400px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <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 />
|<del style='color:#C2DFFF'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</del><br />
|[[Image:pv_BBa_K300015.png|400px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <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 />
|<del style='color:#C2DFFF'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</del><br />
|[[Image:pv_BBa_K300016.png|400px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <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 />
|<del style='color:#C2DFFF'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</del><br />
|[[Image:pv_BBa_K300012.png|400px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <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 />
|'''Self-inducible device''' <br />
| '''Sender Description'''<br />
| '''Receiver Description''' <br />
| '''O.D.start LB''' <br />
| '''K_HSL LB'''<br />
| '''Doubling time LB'''<br />
| '''O.D.start M9''' <br />
| '''K_HSL M9'''<br />
| '''Doubling time M9'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300030.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300028.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300029</partinfo><br> (wiki name: I16)<br> in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300029.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300025.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300026.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300027.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|}<br />
<br />
<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|400px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|[[Image:pv_SwitchPointM9.png|400px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
<br />
<br />
<br />
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=Integrative standard 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). 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). DH5alpha (<partinfo>BBa_V1001</partinfo>) strain was purchased from Invitrogen.<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 as:<br />
<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 />
<br />
'''Pir strains validation:''' in order to test the capability of BW25141 (pir+) and BW23474 (pir-116) to propagate this conditional replication origin, they were made competent, as well as three control strains: MC1061, MG1655 and DH5alpha. 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> (a R6K plasmid with Cm resistance).<br />
<br />
Self-ligated <partinfo>BBa_K300008</partinfo> was prepared by digesting <partinfo>pSB1A2</partinfo>-<partinfo>BBa_K300008</partinfo> (yielded by BioBrick Standard Assembly) with XbaI-SpeI. The insert was isolated and purified from a 1% agarose gel. Then, it was self-ligated to generate a Cm-resistant R6K plasmid. The colonies were counted in each plate and the transformation efficiency was estimated as described before.<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 />
'''BBa_K300000 construction:''' This section describes how this vector backbone was assembled using BioBrick parts.<br />
#'''First step - <partinfo>BBa_K300982</partinfo> intermediate part construction:'''<br />
##<partinfo>BBa_K300983</partinfo>, provided by Mr Gene DNA synthesis service (www.mrgene.com), was excised from its original shipping vector (pMK-RQ) through digestion with AvrII restriction enzyme (Roche). It was then isolated through a 1% agarose gel electrophoresis and gel-extracted (Macherey-Nagel NucleoSpin Extract II).<br />
##<partinfo>BBa_K300008</partinfo> was assembled via BioBrick Standard Assembly from available parts and was excised from its vector (<partinfo>pSB1A2</partinfo>) through digestion with XbaI-SpeI (Roche). It was then isolated using the same procedure described above and it was dephosphorylated by using Antarctic Phosphatase (NEB).<br />
##Digested <partinfo>BBa_K300983</partinfo> and <partinfo>BBa_K300008</partinfo>, all having compatible sticky ends, were ligated with T4 Ligase (Roche) and transformed into competent BW23474 E. coli strain (<partinfo>BBa_K300985</partinfo>) in order to allow the ligated plasmid propagation at high copy number. This strain was necessary because the replication origin of the resulting plasmid was the conditional R6K origin (<partinfo>BBa_J61001</partinfo>).<br />
##Positive transformants, grown on Chloramphenicol (34 ug/ml) plates, were identified by restriction mapping with EcoRI-HindIII (Roche). The yielded plasmid was <partinfo>BBa_K300982</partinfo>.<br />
#'''Second step - <partinfo>BBa_K300000</partinfo> final vector construction:'''<br />
##<partinfo>BBa_K300982</partinfo> DNA was miniprepped, digested with EcoRI-PstI (Roche), run on agarose gel and gel-extracted as described above, in order to cut out and eliminate the RBS (<partinfo>BBa_B0033</partinfo>) between EcoRI and PstI.<br />
##<partinfo>BBa_I52002</partinfo> DNA was excised from <partinfo>pSB4C5</partinfo> by EcoRI-PstI (Roche) digestion and isolated by gel run and gel extraction as described above.<br />
##Digested <partinfo>BBa_K300982</partinfo> and <partinfo>BBa_I52002</partinfo> were ligated and transformed into competent DB3.1 E. coli strain (<partinfo>BBa_V1005</partinfo>), selected with Chloramphenicol at 34 ug/ml.<br />
##Positive transformants were screened by restriction mapping with EcoRI-HindIII (Roche) and sequencing with VF2 (<partinfo>BBa_G00100</partinfo>) and VR (<partinfo>BBa_G00101</partinfo>) standard primers. The yielded plasmid was <partinfo>BBa_K300000</partinfo>.<br />
<br />
All the DNA manipulations were performed according to manufacturer's protocols.<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 />
===Validation of pir strains to propagate the R6K replication origin===<br />
The BW25141 (pir+) and BW23474 (pir-116) ''E. coli'' strains were chosen to propagate the vectors with the R6K replication origin at medium copy (~15 molecules per cell) and high copy (~250) respectively.<br />
The results about their capability to propagate R6K plasmids are shown here:<br />
<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 />
|BW25141 (<partinfo>BBa_K300084</partinfo>, pir+)<br />
|0<br />
|10^5<br />
|10^5<br />
|-<br />
|BW23474 (<partinfo>BBa_K300085</partinfo>, pir-116)<br />
|0<br />
|10^6<br />
|10^6<br />
|-<br />
|DH5alpha (<partinfo>BBa_V1001</partinfo>, neg control)<br />
|0<br />
|10^8<br />
|0<br />
|-<br />
|MC1061 (<partinfo>BBa_K300078</partinfo>, neg control)<br />
|0<br />
|10^6<br />
|0<br />
|-<br />
|MG1655 (<partinfo>BBa_V1000</partinfo>, neg control)<br />
|0<br />
|10^5<br />
|six small colonies from 2 ng of DNA<br />
|}<br />
<br />
<br />
These results show that the R6K conditional 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. The exception was MG1655, for which six small unwanted colonies appeared on the selective plate. Plasmid DNA was purified/HindIII-digested/gel-run for at least one colony for BW25141-<partinfo>BBa_K300008</partinfo> and BW23474-<partinfo>BBa_K300008</partinfo>, while all the six colonies were analyzed for MG1655-<partinfo>BBa_K300008</partinfo> plate. The electrophoresis showed the expected length of the transformed DNA for all the clones except for the MG1655 six colonies, for which a smear was present in the lane (data not shown). Probably, Cm-resistant contaminants were present in the MG1655 culture during the preparation of competent cells.<br />
<br />
For this reason, competent cells were prepared again for MG1655 and the transformation procedure was repeated for this strain, yielding no colonies in the <partinfo>BBa_K300008</partinfo> plate as expected.<br />
<br />
Miniprep of the BW25141 and BW23474 strains transformed with <partinfo>BBa_K300008</partinfo> yielded a DNA concentration of ~20 ng/ul (qualitatively comparable with medium copy number plasmids) and ~90-100 ng/ul (qualitatively comparable with high copy number plasmids).<br />
The results shown in the table above also 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 />
So, the BW25141 and BW23474 strains can be successfully used to propagate the integrative vector after the excision of the pUC19-derived high copy replication origin, present in the default insert <partinfo>BBa_I52002</partinfo>.<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 />
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=Integrative standard vector for yeast=<br />
<br />
==Materials and Methods==<br />
<br />
'''Strain:''' the ''S. cerevisiae'' S288C strain (<partinfo>BBa_K300979</partinfo>, genotype: MATα ρ° trp1-0) was purchased from Open Biosystems.<br />
<br />
'''Construction of BBa_K300001:'''<br />
<br />
#<partinfo>BBa_K300980</partinfo>, provided by Mr Gene DNA synthesis service (www.mrgene.com), was excised from its original shipping vector (pMA) through digestion with MfeI (Fermentas) and NsiI (Fermentas) restriction enzymes. It was then isolated through a 1% agarose gel electrophoresis and gel-extracted (Macherey-Nagel NucleoSpin Extract II).<br />
#<partinfo>BBa_I763007</partinfo>, available in the Registry, was digested with EcoRI-PstI (Roche), ran on agarose gel and its <partinfo>pSB1A2</partinfo> vector was gel-extracted as described above.<br />
#Digested <partinfo>BBa_K300980</partinfo> and <partinfo>pSB1A2</partinfo> have compatible ends (EcoRI-MfeI and PstI-NsiI). They were ligated with T4 Ligase (Roche) and transformed into competent TOP10 E. coli strain (<partinfo>BBa_V1009</partinfo>) which were then plated on Ampicillin (100 ug/ml) plates.<br />
#Positive transformants were identified by colony PCR with VF2 (<partinfo>BBa_G00100</partinfo>) and VR (<partinfo>BBa_G00101</partinfo>) standard primers and by restriction mapping with EcoRI (Roche) or NsiI (Fermentas). The yielded plasmid had <partinfo>BBa_B0033</partinfo> flanked by EcoRI and SpeI.<br />
#The yielded plasmid was then digested with EcoRI-SpeI (Roche) and <partinfo>BBa_K300007</partinfo> (digested with EcoRI-SpeI as well) was assembled in the vector, thus yielding <partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300007</partinfo> (i.e. there is the <partinfo>BBa_K300007</partinfo> part in the cloning site).<br />
<br />
<br />
'''Yeast transformation:'''<br />
<br />
#S288C strain 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 />
==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 />
|6500<br />
|6.5*10^3<br />
|-<br />
|<partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300007</partinfo><br />
|1<br />
|1700<br />
|1.7*10^3<br />
|-<br />
|no DNA<br />
|0<br />
|0<br />
|0<br />
|}<br />
<br />
The colony count was quite difficult (see pictures), but it is useful to have an estimation of the integration efficiency.<br />
<br />
{|align=center<br />
|[[Image:pv_yeastnodna.jpg|thumb|200px|center|S288C transformed with no DNA (negative control).]]<br />
|[[Image:pv_yeast06.jpg|thumb|200px|center|S288C transformed with linearized <partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300006</partinfo>.]]<br />
|[[Image:pv_yeast07.jpg|thumb|200px|center|S288C transformed with linearized <partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300007</partinfo>.]]<br />
|}<br />
<br />
==Discussion==<br />
<br />
The obtained 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 />
Although this integrative vector has already given promising integration results, a lot of work still remains to do:<br />
* the correct phenotype of the S288C bearing these parts has to be validated (by mOrange fluorescence measurement for the <partinfo>BBa_K300007</partinfo> part);<br />
* the actual integration position has to be confirmed by PCR with ad hoc-designed primers;<br />
* the KanMX selection marker excision has to be validated.<br />
<br />
<br />
The compatibility with the BioBrick standard (RFC10, RFC12 and RFC23) enables the engineering the chromosomes of ''S. cerevisiae'' by integrating the desired biological functions following a standard procedure. Moreover, the modularity of the designed integrative vector enables the modification of the integration target site, in fact the default Homologous Regions can only target the Gal genomic system and users may need to target different DNA sequences.<br />
<br />
As reported for the integrative vector for ''E. coli'', the main information about the usage of this integrative vector for yeast is shared in the Registry. We hope to have positively contributed to the diffusion of standard biological parts for yeast, a very interesting chassis for synthetic biology for which only a limited number of parts are available in the Registry and only a few of them has been exaustively characterized.<br />
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=Self-cleaving affinity tags to easily purify proteins=<br />
{{UNIPV-Pavia/Project/results/Self-cleaving_affinity_tags_to_easily_purify_proteins}}<br />
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</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Project/resultsTeam:UNIPV-Pavia/Project/results2010-10-25T10:25:53Z<p>Matteo: /* Materials and Methods */</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="6" align="center" valign="top"><b>Implementation and Results</b></font><br />
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[[#Self-inducible prometers|Self-inducible promoters]]<br />
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[[#Integrative standard vector for E. coli|Integrative standard vector for E. coli]]<br />
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[[#Integrative standard vector for yeast|Integrative standard vector for yeast]]<br />
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[[#Self-cleaving affinity tags to easily purify proteins|Purification of proteins]]<br />
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=Self-inducible promoters=<br />
<br />
===<b>Regulation of signal protein productuion</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> under the regulation of one of these constitutive promoters, we evaluated their 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 R.F.P. in different experimental conditions (plasmids’ copy number and growth medium) :<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 detectable and discernible from the background.<br />
Red Fluorescent Protein (RFP) fluorescence and Optical Density at 600nm (O.D.600) were measured by Tecan Infinite F200 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 DATA Analysis RPU; <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 some 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 some 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 some promoters from Anderson promoters' collection, M9 medium and low copy plasmid (<partinfo>pSB4C5</partinfo>)]]<br />
|}<br />
<br />
'''Discussion''': we observed that the Registry ranking is not always respected. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking of the Registry.<br />
<br />
----<br />
<br />
After the evaluation of promoters’ activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream 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 />
<br />
Some of the promoters probably induce a production of LuxI that is injurious for ''E. coli'', so it wasn’t possible to exploit all the combinations. <br />
For every part, a measurement system was built, exploiting the production of the reporter gene GFP to evaluate the trascription initiation point for every promoter. Many different combination were explored, in order to provide a library of promoters capable of 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> sensor was contained in <partinfo>pSB1A3</partinfo> in ''E. coli'' TOP10.<br />
<br />
'''Results''' The amount of 3OC6-HSL produced after 6 hours growth by ''E. coli'' DH5alpha bearing the parts contained in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Figure 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 />
{| <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 Figure 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 />
{| <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 generator to produce 3OC6-HSL signal molecule. Data are partially uncompleted because for weak promoters ore medium-strength promoters contained in low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. This simple experiment shows that there's a strong correlation between the strength of promoter and the amount of signal molecule produced, even if the correspondence is not exact, probably due to the fact that very strong promoters (such as <partinfo>BBa_J23101</partinfo>) give to the cells a high metabolic burden, thus resulting in lower production yields.<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 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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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>in <partinfo>pSB1A2</partinfo><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 />
Doubling times were estimated as explained [[Team:UNIPV-Pavia/Parts/Characterization#Doubling time evaluation|here]]<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, as reported [[Team:UNIPV-Pavia/Parts/Characterization#Data analysis for self-inducible promoters (initiation-treshold determination)| in this section]]. Results are summarized in the following tables:<br />
<br />
<br />
<br />
<br />
'''Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>'''<br />
<br />
{| border='1'<br />
|'''Self-inducible device''' || '''Description''' || '''O.D.start LB''' || '''K_HSL LB'''|| '''Doubling time LB'''|| '''O.D.start M9''' || '''K_HSL M9'''|| '''Doubling time M9'''<br />
|-<br />
|<partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid <br />
|[[Image:pv_BBa_K300017.png|400px]]<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid<br />
|[[Image:pv_BBa_K300014.png|400px]]<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid<br />
|[[Image:pv_BBa_K300015.png|400px]]<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid<br />
|[[Image:pv_BBa_K300016.png|400px]]<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid<br />
|[[Image:pv_BBa_K300012.png|400px]]<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|}<br />
<br />
'''Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>'''<br />
{| border='1'<br />
|'''Self-inducible device''' || '''Description''' || '''O.D.start LB''' || '''K_HSL LB'''|| '''Doubling time LB'''|| '''O.D.start M9''' || '''K_HSL M9'''|| '''Doubling time M9'''<br />
|-<br />
|<partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB1A2</partinfo> plasmid <br />
|[[Image:pv_BBa_K300017.png|400px]]<br />
| <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 />
|-<br />
|<del style='color:#C2DFFF'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB1A2</partinfo> plasmid</del><br />
|[[Image:pv_BBa_K300014.png|400px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <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 />
|<del style='color:#C2DFFF'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB1A2</partinfo> plasmid</del><br />
|[[Image:pv_BBa_K300015.png|400px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <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 />
|<del style='color:#C2DFFF'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB1A2</partinfo> plasmid</del><br />
|[[Image:pv_BBa_K300016.png|400px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <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 />
|<del style='color:#C2DFFF'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB1A2</partinfo> plasmid</del><br />
|[[Image:pv_BBa_K300012.png|400px]]<br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <font color='red' size='+2'>X</font><br />
| <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 />
|'''Self-inducible device''' <br />
| '''Sender Description'''<br />
| '''Receiver Description''' <br />
| '''O.D.start LB''' <br />
| '''K_HSL LB'''<br />
| '''Doubling time LB'''<br />
| '''O.D.start M9''' <br />
| '''K_HSL M9'''<br />
| '''Doubling time M9'''<br />
|-<br />
|<partinfo>BBa_K300030</partinfo><br> (wiki name: I14) <br>in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300030.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300028</partinfo><br> (wiki name: I15)<br> in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300028.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300029</partinfo><br> (wiki name: I16)<br> in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300029.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300025</partinfo><br> (wiki name: I17) <br>in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300025.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300026</partinfo><br> (wiki name: I18) <br>in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300026.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|-<br />
|<partinfo>BBa_K300027</partinfo><br> (wiki name: I19)<br> in <partinfo>pSB4C5</partinfo> plasmid <br />
|[[Image:pv_BBa_K300027.png|170px]]<br>LC<br />
|[[Image:pv_BBa_F2620.png|170px]]<br>HC<br />
| <br />
| <br />
| <br />
|<br />
|<br />
|<br />
|}<br />
<br />
<br />
{|<br />
|[[Image:pv_SwitchPointLB.png|400px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in LB]]<br />
|[[Image:pv_SwitchPointM9.png|400px|thumb|center| Average growth curve with ODstart evaluated by Threshold algorithm in M9]]<br />
|}<br />
<br />
<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/UNIPV_Pavia_Icona_intColi.GIF" width="55px" height="70px"/><br />
</html><br />
<br />
=Integrative standard 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). 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). DH5alpha (<partinfo>BBa_V1001</partinfo>) strain was purchased from Invitrogen.<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 the figure below:<br />
<br />
{|align=center<br />
|[[Image:relativeprimers.png|thumb|450px|center|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 as:<br />
<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 />
<br />
'''Pir strains validation:''' in order to test the capability of BW25141 (pir+) and BW23474 (pir-116) to propagate this conditional replication origin, they were made competent, as well as three control strains: MC1061, MG1655 and DH5alpha. 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> (a R6K plasmid with Cm resistance).<br />
<br />
Self-ligated <partinfo>BBa_K300008</partinfo> was prepared by digesting <partinfo>pSB1A2</partinfo>-<partinfo>BBa_K300008</partinfo> (yielded by BioBrick Standard Assembly) with XbaI-SpeI. The insert was isolated and purified from a 1% agarose gel. Then, it was self-ligated to generate a Cm-resistant R6K plasmid. The colonies were counted in each plate and the transformation efficiency was estimated as described before.<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 />
'''BBa_K300000 construction:''' This section describes how this vector backbone was assembled using BioBrick parts.<br />
#'''First step - <partinfo>BBa_K300982</partinfo> intermediate part construction:'''<br />
##<partinfo>BBa_K300983</partinfo>, provided by Mr Gene DNA synthesis service (www.mrgene.com), was excised from its original shipping vector (pMK-RQ) through digestion with AvrII restriction enzyme (Roche). It was then isolated through a 1% agarose gel electrophoresis and gel-extracted (Macherey-Nagel NucleoSpin Extract II).<br />
##<partinfo>BBa_K300008</partinfo> was assembled via BioBrick Standard Assembly from available parts and was excised from its vector (<partinfo>pSB1A2</partinfo>) through digestion with XbaI-SpeI (Roche). It was then isolated using the same procedure described above and it was dephosphorylated by using Antarctic Phosphatase (NEB).<br />
##Digested <partinfo>BBa_K300983</partinfo> and <partinfo>BBa_K300008</partinfo>, all having compatible sticky ends, were ligated with T4 Ligase (Roche) and transformed into competent BW23474 E. coli strain (<partinfo>BBa_K300985</partinfo>) in order to allow the ligated plasmid propagation at high copy number. This strain was necessary because the replication origin of the resulting plasmid was the conditional R6K origin (<partinfo>BBa_J61001</partinfo>).<br />
##Positive transformants, grown on Chloramphenicol (34 ug/ml) plates, were identified by restriction mapping with EcoRI-HindIII (Roche). The yielded plasmid was <partinfo>BBa_K300982</partinfo>.<br />
#'''Second step - <partinfo>BBa_K300000</partinfo> final vector construction:'''<br />
##<partinfo>BBa_K300982</partinfo> DNA was miniprepped, digested with EcoRI-PstI (Roche), run on agarose gel and gel-extracted as described above, in order to cut out and eliminate the RBS (<partinfo>BBa_B0033</partinfo>) between EcoRI and PstI.<br />
##<partinfo>BBa_I52002</partinfo> DNA was excised from <partinfo>pSB4C5</partinfo> by EcoRI-PstI (Roche) digestion and isolated by gel run and gel extraction as described above.<br />
##Digested <partinfo>BBa_K300982</partinfo> and <partinfo>BBa_I52002</partinfo> were ligated and transformed into competent DB3.1 E. coli strain (<partinfo>BBa_V1005</partinfo>), selected with Chloramphenicol at 34 ug/ml.<br />
##Positive transformants were screened by restriction mapping with EcoRI-HindIII (Roche) and sequencing with VF2 (<partinfo>BBa_G00100</partinfo>) and VR (<partinfo>BBa_G00101</partinfo>) standard primers. The yielded plasmid was <partinfo>BBa_K300000</partinfo>.<br />
<br />
All the DNA manipulations were performed according to manufacturer's protocols.<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 />
===Validation of pir strains to propagate the R6K replication origin===<br />
The BW25141 (pir+) and BW23474 (pir-116) ''E. coli'' strains were chosen to propagate the vectors with the R6K replication origin at medium copy (~15 molecules per cell) and high copy (~250) respectively.<br />
The results about their capability to propagate R6K plasmids are shown here:<br />
<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 />
|BW25141 (<partinfo>BBa_K300084</partinfo>, pir+)<br />
|0<br />
|10^5<br />
|10^5<br />
|-<br />
|BW23474 (<partinfo>BBa_K300085</partinfo>, pir-116)<br />
|0<br />
|10^6<br />
|10^6<br />
|-<br />
|DH5alpha (<partinfo>BBa_V1001</partinfo>, neg control)<br />
|0<br />
|10^8<br />
|0<br />
|-<br />
|MC1061 (<partinfo>BBa_K300078</partinfo>, neg control)<br />
|0<br />
|10^6<br />
|0<br />
|-<br />
|MG1655 (<partinfo>BBa_V1000</partinfo>, neg control)<br />
|0<br />
|10^5<br />
|six small colonies from 2 ng of DNA<br />
|}<br />
<br />
<br />
These results show that the R6K conditional 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. The exception was MG1655, for which six small unwanted colonies appeared on the selective plate. Plasmid DNA was purified/HindIII-digested/gel-run for at least one colony for BW25141-<partinfo>BBa_K300008</partinfo> and BW23474-<partinfo>BBa_K300008</partinfo>, while all the six colonies were analyzed for MG1655-<partinfo>BBa_K300008</partinfo> plate. The electrophoresis showed the expected length of the transformed DNA for all the clones except for the MG1655 six colonies, for which a smear was present in the lane (data not shown). Probably, Cm-resistant contaminants were present in the MG1655 culture during the preparation of competent cells.<br />
<br />
For this reason, competent cells were prepared again for MG1655 and the transformation procedure was repeated for this strain, yielding no colonies in the <partinfo>BBa_K300008</partinfo> plate as expected.<br />
<br />
Miniprep of the BW25141 and BW23474 strains transformed with <partinfo>BBa_K300008</partinfo> yielded a DNA concentration of ~20 ng/ul (qualitatively comparable with medium copy number plasmids) and ~90-100 ng/ul (qualitatively comparable with high copy number plasmids).<br />
The results shown in the table above also 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 />
So, the BW25141 and BW23474 strains can be successfully used to propagate the integrative vector after the excision of the pUC19-derived high copy replication origin, present in the default insert <partinfo>BBa_I52002</partinfo>.<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 />
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'''<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.<br />
<br />
PCR results with primers VF2/VR showed that each clone had<br />
<br />
<br />
<br />
'''Validation of the marker-less phenotype:'''<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 />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/0/03/UNIPV_Pavia_Icona_IntYeast.GIF" width="55px" height="70px"/><br />
</html><br />
<br />
=Integrative standard vector for yeast=<br />
<br />
==Materials and Methods==<br />
<br />
'''Strain:''' the ''S. cerevisiae'' S288C strain (<partinfo>BBa_K300979</partinfo>, genotype: MATα ρ° trp1-0) was purchased from Open Biosystems.<br />
<br />
'''Construction of BBa_K300001:'''<br />
<br />
#<partinfo>BBa_K300980</partinfo>, provided by Mr Gene DNA synthesis service (www.mrgene.com), was excised from its original shipping vector (pMA) through digestion with MfeI (Fermentas) and NsiI (Fermentas) restriction enzymes. It was then isolated through a 1% agarose gel electrophoresis and gel-extracted (Macherey-Nagel NucleoSpin Extract II).<br />
#<partinfo>BBa_I763007</partinfo>, available in the Registry, was digested with EcoRI-PstI (Roche), ran on agarose gel and its <partinfo>pSB1A2</partinfo> vector was gel-extracted as described above.<br />
#Digested <partinfo>BBa_K300980</partinfo> and <partinfo>pSB1A2</partinfo> have compatible ends (EcoRI-MfeI and PstI-NsiI). They were ligated with T4 Ligase (Roche) and transformed into competent TOP10 E. coli strain (<partinfo>BBa_V1009</partinfo>) which were then plated on Ampicillin (100 ug/ml) plates.<br />
#Positive transformants were identified by colony PCR with VF2 (<partinfo>BBa_G00100</partinfo>) and VR (<partinfo>BBa_G00101</partinfo>) standard primers and by restriction mapping with EcoRI (Roche) or NsiI (Fermentas). The yielded plasmid had <partinfo>BBa_B0033</partinfo> flanked by EcoRI and SpeI.<br />
#The yielded plasmid was then digested with EcoRI-SpeI (Roche) and <partinfo>BBa_K300007</partinfo> (digested with EcoRI-SpeI as well) was assembled in the vector, thus yielding <partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300007</partinfo> (i.e. there is the <partinfo>BBa_K300007</partinfo> part in the cloning site).<br />
<br />
<br />
'''Yeast transformation:'''<br />
<br />
#S288C strain 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 0.5-1 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 />
==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 />
|???<br />
|???<br />
|???<br />
|-<br />
|<partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300007</partinfo><br />
|???<br />
|???<br />
|???<br />
|-<br />
|no DNA<br />
|0<br />
|0<br />
|0<br />
|}<br />
<br />
The colony count was quite difficult (see pictures), but it is useful to have an estimation of the integration efficiency.<br />
<br />
{|align=center<br />
|[[Image:pv_yeastnodna.jpg|thumb|200px|center|S288C transformed with no DNA (negative control).]]<br />
|[[Image:pv_yeast06.jpg|thumb|200px|center|S288C transformed with linearized <partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300006</partinfo>.]]<br />
|[[Image:pv_yeast07.jpg|thumb|200px|center|S288C transformed with linearized <partinfo>BBa_K300001</partinfo>-<partinfo>BBa_K300007</partinfo>.]]<br />
|}<br />
<br />
==Discussion==<br />
<br />
The obtained 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 />
Although this integrative vector has already given promising integration results, a lot of work still remains to do:<br />
* the correct phenotype of the S288C bearing these parts has to be validated (by mOrange fluorescence measurement for the <partinfo>BBa_K300007</partinfo> part);<br />
* the actual integration position has to be confirmed by PCR with ad hoc-designed primers;<br />
* the KanMX selection marker excision has to be validated.<br />
<br />
<br />
The compatibility with the BioBrick standard (RFC10, RFC12 and RFC23) enables the engineering the chromosomes of ''S. cerevisiae'' by integrating the desired biological functions following a standard procedure. Moreover, the modularity of the designed integrative vector enables the modification of the integration target site, in fact the default Homologous Regions can only target the Gal genomic system and users may need to target different DNA sequences.<br />
<br />
As reported for the integrative vector for ''E. coli'', the main information about the usage of this integrative vector for yeast is shared in the Registry. We hope to have positively contributed to the diffusion of standard biological parts for yeast, a very interesting chassis for synthetic biology for which only a limited number of parts are available in the Registry and only a few of them has been exaustively characterized.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<html><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 />
=Self-cleaving affinity tags to easily purify proteins=<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==PHB production==<br />
<br />
<br />
==Fusion protein synthesis==<br />
<br />
==Phasins binding activity==<br />
<br />
<br />
<br />
</td></tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Team/AdvisorsTeam:UNIPV-Pavia/Team/Advisors2010-10-25T10:17:18Z<p>Matteo: /* Matteo Meroso */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="3"> {{UNIPV-Pavia/header}} </td> <br />
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<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<!-- Contenuti --><br />
<br />
<td valign="top"><br />
<table border="0" align="center" ><tr><td align="left" valign="top" colspan="3" style="padding:20px"><br />
<html><p align="center"><font size="4"><b>ADVISORS</b></font></p></html><hr><br><br />
<br />
== Manuel Lupotto ==<br />
<br />
<table><tr><br />
<td width="20%" valign='top'><br />
{| border="0"<br />
| [[Image:UNIPV_Pavia_manuellupotto1.jpg | 200px |left|Manuel Lupotto]]<br />
|}<br />
<br />
</td><br />
<td valign='top'><br />
<br />
*'''Academic'''<br />
Bachelor Degree in Biology at Università degli Studi di Pavia <br />
<br />
*'''Contacts'''<br />
manuel7d9[AT]gmail[DOT]com<br />
</td><br />
<br />
</tr><br />
</table><br />
<br><br />
<br />
== Matteo Meroso ==<br />
<br />
<table><tr><td valign='top'><br />
<br />
*'''Academic'''<br />
Bachelor Degree in Biomedical Engineering (2007) and now Master student in Biomedical Engineering at Università degli Studi di Pavia<br />
<br />
*'''Personal''' <br />
Hi guys!! I'm Matteo and I'm 25 years old. I come from Voghera, a small town close to Pavia.<br />
<br><br />
I'm overjoyed to take part in iGEM competition because I expect to increase my skill to work in team and improve my knowledge in synthetic biology!!!<br />
<br><br />
See you at the Jamboree!!!!<br />
<br><br />
<br />
*'''Contacts''' <br />
matteo[DOT]meroso01[AT]libero[DOT]it<br />
</td><br />
<td width="20%" valign='top'><br />
{| border="0"<br />
| [[Image:UNIPV_Pavia_matteomeroso.JPG| 200px |right|Matteo Meroso]]<br />
|}<br />
<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
== Lorenzo Pasotti ==<br />
<table><tr><br />
<br />
<td width="20%" valign='top'><br />
{| border="0"<br />
| [[Image:UNIPV_Pavia_lorenzopasotti.jpg | 200px |right|Lorenzo Pasotti]]<br />
|}<br />
<br />
</td><br />
<br />
<td valign='top'><br />
<br />
*'''Academic'''<br />
PhD student in Bioengineering and Bioinformatics at the Università degli Studi di Pavia, working on Synthetic Biology in the Lab for Biomedical Informatics and in the Centre for Tissue Engineering.<br />
<br />
*'''Contact'''<br />
lorenzo[DOT]pasotti[AT]unipv[DOT]it<br />
</td><br />
<br />
</tr><br />
</table><br />
<br><br />
<br />
== Giacomo Zambianchi ==<br />
<br />
<table><tr><td valign='top'><br />
<br />
*'''Academic'''<br />
I'm finishing my studies in Biomedical Engineering and in September 2007 I took the Bachelor Degree (always in Biomedical Engineering) at Università degli Studi di Pavia.<br />
<br />
*'''Personal'''<br />
<html><br />
Hi and nice to meet you again,<br />
I'm Giacomo, 25 years old, and if you remember me it's because last year I already took part in iGEM - this competition addicted me - as student. This year I'm advisor of the brand new team from Pavia and we are even more competitive than the last one!<br />
</html><br />
<br />
*'''Contacts''': <br />
giacomo[DOT]zambianchi[AT]virgilio[DOT]it<br />
<br />
</td><br />
<td width="20%" valign='top'><br />
{| border="0"<br />
| [[Image:UNIPV_Pavia_giacomozambianchi.jpg | 250px |right|Giacomo Zambianchi]]<br />
|}<br />
<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
== Susanna Zucca ==<br />
<br />
<table><tr><br />
<td width="20%" valign='top'><br />
{| border="0"<br />
| [[Image:UNIPV_Pavia_susannazucca.jpg | 200px |left|Susanna Zucca]]<br />
|}<br />
<br />
</td><br />
<td valign='top'><br />
<br />
*'''Academic'''<br />
Bachelor Degree in Biomedical Engineering and Master Student in Biomedical Engineering at Università degli Studi di Pavia<br />
<br />
*'''Personal'''<br />
<html><br />
I was born on November 14th, 1985 in Pavia. <br />
I think that iGEM experience is terrific, for it gives the opportunity to meet new people and to enjoy a beautiful experience, both coltural and personal. <br />
</html><br />
<br />
*'''Contacts''': <br />
susanna[DOT]zucca[AT]live[DOT]it<br />
<br />
</td><br />
<br />
</tr><br />
</table><br />
<br />
<br />
<br />
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<font class="menu"><br />
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[https://2010.igem.org/Team:UNIPV-Pavia/Team Team]<br />
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[https://2010.igem.org/Team:UNIPV-Pavia/Team/Instructors Instructors]<br />
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[https://2010.igem.org/Team:UNIPV-Pavia/Team/Advisors Advisors]<br />
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[https://2010.igem.org/Team:UNIPV-Pavia/Team/Students Students]<br />
</td></tr></div>Matteohttp://2010.igem.org/File:UNIPV10_19_10_10_MC42ABC_MC43ABC_MG42BC_MG43ABC_BLANK_MC1061_MG1655(TUTTI_P3-P4).jpgFile:UNIPV10 19 10 10 MC42ABC MC43ABC MG42BC MG43ABC BLANK MC1061 MG1655(TUTTI P3-P4).jpg2010-10-25T10:11:28Z<p>Matteo: </p>
<hr />
<div></div>Matteohttp://2010.igem.org/File:UNIPV10_19_10_10_MC42ABC_MC43ABC_MG42BC_MG43ABC_BLANK_MC1061_MG1655(TUTTI_P2-P3).jpgFile:UNIPV10 19 10 10 MC42ABC MC43ABC MG42BC MG43ABC BLANK MC1061 MG1655(TUTTI P2-P3).jpg2010-10-25T10:10:52Z<p>Matteo: </p>
<hr />
<div></div>Matteohttp://2010.igem.org/File:UNIPV10_19_10_10_MC42ABC_MC43ABC_MG42BC_MG43ABC_BLANK_MC1061_MG1655(TUTTI_P1-P2).jpgFile:UNIPV10 19 10 10 MC42ABC MC43ABC MG42BC MG43ABC BLANK MC1061 MG1655(TUTTI P1-P2).jpg2010-10-25T10:10:14Z<p>Matteo: </p>
<hr />
<div></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana3Team:UNIPV-Pavia/Calendar/October/settimana32010-10-25T10:09:42Z<p>Matteo: /* October, 19th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
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{{UNIPV-Pavia/Style}}<br />
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<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
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<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 3</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 18th==<br />
<br />
We have finally completed the parts transfer to <partinfo>pSB1C3</partinfo> plasmid.<br />
All the parts that will be sent to the registry were previously screened by UNIPV-Pavia TEAM, as reported in this notebook and all of them were sequenced and sequencing results were correct.<br />
<br />
After parts have been moved to the shipping plasmid and checked by agarose gel electrophoresis.<br />
<br />
The parts we sent to MIT and submitted to the registry are:<br />
<br />
<table border='1' align='center'><br />
<tr><th>Part name</th><th>Wiki name</th><th>Plasmid</th><th>Number</th></tr><br />
<tr><td><partinfo>BBa_K300005</partinfo></td><td>I0</td><td>pSB1C3</td><td>1</td></tr><br />
<tr><td><partinfo>BBa_K300006</partinfo></td><td>I1</td><td>pSB1C3</td><td>2</td></tr><br />
<tr><td><partinfo>BBa_K300009</partinfo></td><td>I3</td><td>pSB1C3</td><td>3</td></tr><br />
<tr><td><partinfo>BBa_K300008</partinfo></td><td>I5</td><td>pSB1C3</td><td>4</td></tr><br />
<tr><td><partinfo>BBa_K300024</partinfo></td><td>I7</td><td>pSB1C3</td><td>5</td></tr><br />
<tr><td><partinfo>BBa_K300019</partinfo></td><td>I12</td><td>pSB1C3</td><td>6</td></tr><br />
<tr><td><partinfo>BBa_K300030</partinfo></td><td>I14</td><td>pSB1C3</td><td>7</td></tr><br />
<tr><td><partinfo>BBa_K300028</partinfo></td><td>I15</td><td>pSB1C3</td><td>8</td></tr><br />
<tr><td><partinfo>BBa_K300029</partinfo></td><td>I16</td><td>pSB1C3</td><td>9</td></tr><br />
<tr><td><partinfo>BBa_K300025</partinfo></td><td>I17</td><td>pSB1C3</td><td>10</td></tr><br />
<tr><td><partinfo>BBa_K300026</partinfo></td><td>I18</td><td>pSB1C3</td><td>11</td></tr><br />
<tr><td><partinfo>BBa_K300027</partinfo></td><td>I19</td><td>pSB1C3</td><td>12</td></tr><br />
<tr><td><partinfo>BBa_K300002</partinfo></td><td>I20</td><td>pSB1C3</td><td>13</td></tr><br />
<tr><td><partinfo>BBa_K300003</partinfo></td><td>I21</td><td>pSB1C3</td><td>14</td></tr><br />
<tr><td><partinfo>BBa_K300031</partinfo></td><td>I22</td><td>pSB1C3</td><td>15</td></tr><br />
<tr><td><partinfo>BBa_K300081</partinfo></td><td>I26</td><td>pSB1C3</td><td>16</td></tr><br />
<tr><td><partinfo>BBa_K300079</partinfo></td><td>I31</td><td>pSB1C3</td><td>17</td></tr><br />
<tr><td><partinfo>BBa_K300080</partinfo></td><td>I35</td><td>pSB1C3</td><td>18</td></tr><br />
<tr><td><partinfo>BBa_K300086</partinfo></td><td>I47</td><td>pSB1C3</td><td>19</td></tr><br />
<tr><td><partinfo>BBa_K300088</partinfo></td><td>I48</td><td>pSB1C3</td><td>20</td></tr><br />
<tr><td><partinfo>BBa_K300090</partinfo></td><td>I49</td><td>pSB1C3</td><td>21</td></tr><br />
<tr><td><partinfo>BBa_K300082</partinfo></td><td>I55</td><td>pSB1C3</td><td>22</td></tr><br />
<tr><td><partinfo>BBa_K300084</partinfo></td><td>I78</td><td>pSB1C3</td><td>23</td></tr><br />
<tr><td><partinfo>BBa_K300083</partinfo></td><td>I79</td><td>pSB1C3</td><td>24</td></tr><br />
<tr><td><partinfo>BBa_K300010</partinfo></td><td>I80</td><td>pSB1C3</td><td>25</td></tr><br />
<tr><td><partinfo>BBa_K300007</partinfo></td><td>I70 - BioBrick</td><td><partinfo>BBa_K300001</partinfo></td><td>26</td></tr><br />
<tr><td><partinfo>BBa_K300007</partinfo></td><td>I70 - vector</td><td><partinfo>BBa_K300001</partinfo></td><td>27</td></tr><br />
<tr><td><partinfo>BBa_K300004</partinfo></td><td>INTEIN</td><td>pSB1C3</td><td>28</td></tr><br />
<tr><td><partinfo>BBa_I52002</partinfo></td><td>I51</td><td><partinfo>BBa_K300000</partinfo></td><td>29</td></tr><br />
</table><br />
<br />
<br />
The parts we have in freezer and we only submitted to the Registry are:<br />
<table border='1' align='center'><br />
<tr><th>Part name</th><th>Wiki name</th><th>Plasmid</th></tr><br />
<tr><td><partinfo>BBa_K300021</partinfo></td><td>I8</td><td><partinfo>BBa_J61002</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300022</partinfo></td><td>I9</td><td><partinfo>BBa_J61002</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300023</partinfo></td><td>I10</td><td><partinfo>BBa_J61002</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300091</partinfo></td><td>I63</td><td><partinfo>pSB1AK3</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300092</partinfo></td><td>I64</td><td><partinfo>pSB1A2</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300099</partinfo></td><td>I65</td><td><partinfo>pSB1A2</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300980</partinfo></td><td>YEAST MR.G</td><td>pMA commercial vector</td></tr><br />
<tr><td><partinfo>BBa_K300982</partinfo></td><td>CREAM</td><td>-</td></tr><br />
<tr><td><partinfo>BBa_K300983</partinfo></td><td>CRIM MR.G</td><td>pMK-RQ commercial vector</td></tr><br />
<!--tr><td><partinfo></partinfo></td><td></td><td><partinfo></partinfo></td></tr--><br />
</table><br />
<br />
<br />
----<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 19th==<br />
<b>Wiki update and definition of results.</b><br />
<br />
----<br />
Every clones of E. coli genome integration project were amplified with PCR to verify their lengths and their positions into the genome.<br />
In order to obtain better results we perform all the amplifications using an annealing temperature of 63°C.<br />
Screening PCR was performed on MC42(A-B-C), MC43(A-B-C), MG42(B-C), MG43(A-B-C), MC1061 and MG1655. Primers used were BBa_K300975-BBa_K300976 (P1-P2), BBa_K300976-BBa_K300977 (P2-P3), BBa_K300977-BBa_K300978 (P3-P4).<br />
<br />
[[Image:UNIPV10_19_10_10_MC42ABC_MC43ABC_MG42BC_MG43ABC_BLANK_MC1061_MG1655(TUTTI P1-P2).jpg|thumb|300px|center|The order of the samples was the follow: MC42(A,B,C), MC43(A,B,C), MG42(B,C), MG43(A,B,C), blank, MC1061, MG1655. Primers were BBa_K300975-BBa_K300976 (P1-P2).]]<br />
<br />
[[Image:UNIPV10_19_10_10_MC42ABC_MC43ABC_MG42BC_MG43ABC_BLANK_MC1061_MG1655(TUTTI P2-P3).jpg|thumb|300px|center|The order of the samples was the follow: MC42(A,B,C), MC43(A,B,C), MG42(B,C), MG43(A,B,C), blank, MC1061, MG1655. Primers were BBa_K300976-BBa_K300977 (P2-P3).]]<br />
<br />
[[Image:UNIPV10_19_10_10_MC42ABC_MC43ABC_MG42BC_MG43ABC_BLANK_MC1061_MG1655(TUTTI P3-P4).jpg|thumb|300px|center|The order of the samples was the follow: MC42(A,B,C), MC43(A,B,C), MG42(B,C), MG43(A,B,C), blank, MC1061, MG1655. Primers were BBa_K300977-BBa_K300978 (P3-P4).]]<br />
<br />
<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 20th==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 21st==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 22nd==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 23rd==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
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<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana2| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana4| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana3Team:UNIPV-Pavia/Calendar/October/settimana32010-10-25T10:01:44Z<p>Matteo: /* October, 18th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 3</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 18th==<br />
<br />
We have finally completed the parts transfer to <partinfo>pSB1C3</partinfo> plasmid.<br />
All the parts that will be sent to the registry were previously screened by UNIPV-Pavia TEAM, as reported in this notebook and all of them were sequenced and sequencing results were correct.<br />
<br />
After parts have been moved to the shipping plasmid and checked by agarose gel electrophoresis.<br />
<br />
The parts we sent to MIT and submitted to the registry are:<br />
<br />
<table border='1' align='center'><br />
<tr><th>Part name</th><th>Wiki name</th><th>Plasmid</th><th>Number</th></tr><br />
<tr><td><partinfo>BBa_K300005</partinfo></td><td>I0</td><td>pSB1C3</td><td>1</td></tr><br />
<tr><td><partinfo>BBa_K300006</partinfo></td><td>I1</td><td>pSB1C3</td><td>2</td></tr><br />
<tr><td><partinfo>BBa_K300009</partinfo></td><td>I3</td><td>pSB1C3</td><td>3</td></tr><br />
<tr><td><partinfo>BBa_K300008</partinfo></td><td>I5</td><td>pSB1C3</td><td>4</td></tr><br />
<tr><td><partinfo>BBa_K300024</partinfo></td><td>I7</td><td>pSB1C3</td><td>5</td></tr><br />
<tr><td><partinfo>BBa_K300019</partinfo></td><td>I12</td><td>pSB1C3</td><td>6</td></tr><br />
<tr><td><partinfo>BBa_K300030</partinfo></td><td>I14</td><td>pSB1C3</td><td>7</td></tr><br />
<tr><td><partinfo>BBa_K300028</partinfo></td><td>I15</td><td>pSB1C3</td><td>8</td></tr><br />
<tr><td><partinfo>BBa_K300029</partinfo></td><td>I16</td><td>pSB1C3</td><td>9</td></tr><br />
<tr><td><partinfo>BBa_K300025</partinfo></td><td>I17</td><td>pSB1C3</td><td>10</td></tr><br />
<tr><td><partinfo>BBa_K300026</partinfo></td><td>I18</td><td>pSB1C3</td><td>11</td></tr><br />
<tr><td><partinfo>BBa_K300027</partinfo></td><td>I19</td><td>pSB1C3</td><td>12</td></tr><br />
<tr><td><partinfo>BBa_K300002</partinfo></td><td>I20</td><td>pSB1C3</td><td>13</td></tr><br />
<tr><td><partinfo>BBa_K300003</partinfo></td><td>I21</td><td>pSB1C3</td><td>14</td></tr><br />
<tr><td><partinfo>BBa_K300031</partinfo></td><td>I22</td><td>pSB1C3</td><td>15</td></tr><br />
<tr><td><partinfo>BBa_K300081</partinfo></td><td>I26</td><td>pSB1C3</td><td>16</td></tr><br />
<tr><td><partinfo>BBa_K300079</partinfo></td><td>I31</td><td>pSB1C3</td><td>17</td></tr><br />
<tr><td><partinfo>BBa_K300080</partinfo></td><td>I35</td><td>pSB1C3</td><td>18</td></tr><br />
<tr><td><partinfo>BBa_K300086</partinfo></td><td>I47</td><td>pSB1C3</td><td>19</td></tr><br />
<tr><td><partinfo>BBa_K300088</partinfo></td><td>I48</td><td>pSB1C3</td><td>20</td></tr><br />
<tr><td><partinfo>BBa_K300090</partinfo></td><td>I49</td><td>pSB1C3</td><td>21</td></tr><br />
<tr><td><partinfo>BBa_K300082</partinfo></td><td>I55</td><td>pSB1C3</td><td>22</td></tr><br />
<tr><td><partinfo>BBa_K300084</partinfo></td><td>I78</td><td>pSB1C3</td><td>23</td></tr><br />
<tr><td><partinfo>BBa_K300083</partinfo></td><td>I79</td><td>pSB1C3</td><td>24</td></tr><br />
<tr><td><partinfo>BBa_K300010</partinfo></td><td>I80</td><td>pSB1C3</td><td>25</td></tr><br />
<tr><td><partinfo>BBa_K300007</partinfo></td><td>I70 - BioBrick</td><td><partinfo>BBa_K300001</partinfo></td><td>26</td></tr><br />
<tr><td><partinfo>BBa_K300007</partinfo></td><td>I70 - vector</td><td><partinfo>BBa_K300001</partinfo></td><td>27</td></tr><br />
<tr><td><partinfo>BBa_K300004</partinfo></td><td>INTEIN</td><td>pSB1C3</td><td>28</td></tr><br />
<tr><td><partinfo>BBa_I52002</partinfo></td><td>I51</td><td><partinfo>BBa_K300000</partinfo></td><td>29</td></tr><br />
</table><br />
<br />
<br />
The parts we have in freezer and we only submitted to the Registry are:<br />
<table border='1' align='center'><br />
<tr><th>Part name</th><th>Wiki name</th><th>Plasmid</th></tr><br />
<tr><td><partinfo>BBa_K300021</partinfo></td><td>I8</td><td><partinfo>BBa_J61002</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300022</partinfo></td><td>I9</td><td><partinfo>BBa_J61002</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300023</partinfo></td><td>I10</td><td><partinfo>BBa_J61002</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300091</partinfo></td><td>I63</td><td><partinfo>pSB1AK3</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300092</partinfo></td><td>I64</td><td><partinfo>pSB1A2</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300099</partinfo></td><td>I65</td><td><partinfo>pSB1A2</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300980</partinfo></td><td>YEAST MR.G</td><td>pMA commercial vector</td></tr><br />
<tr><td><partinfo>BBa_K300982</partinfo></td><td>CREAM</td><td>-</td></tr><br />
<tr><td><partinfo>BBa_K300983</partinfo></td><td>CRIM MR.G</td><td>pMK-RQ commercial vector</td></tr><br />
<!--tr><td><partinfo></partinfo></td><td></td><td><partinfo></partinfo></td></tr--><br />
</table><br />
<br />
<br />
----<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 19th==<br />
<b>Wiki update and definition of results.</b><br />
<br />
----<br />
Every clones of E. coli genome integration project were amplified with PCR to verify their lengths and their positions into the genome.<br />
<br />
[[Image:UNIPV10_19_10_10_MC42ABC_MC43ABC_MG42BC_MG43ABC_BLANK_MC1061_MG1655(TUTTI P1-P2).jpg|thumb|300px|center|The order of the samples was the follow: MC42(A,B,C), MC43(A,B,C), MG42(B,C), MG43(A,B,C), blank, MC1061, MG1655. Primers were BBa_K300975-BBa_K300976 (P1-P2).]]<br />
<br />
[[Image:UNIPV10_19_10_10_MC42ABC_MC43ABC_MG42BC_MG43ABC_BLANK_MC1061_MG1655(TUTTI P2-P3).jpg|thumb|300px|center|The order of the samples was the follow: MC42(A,B,C), MC43(A,B,C), MG42(B,C), MG43(A,B,C), blank, MC1061, MG1655. Primers were BBa_K300976-BBa_K300977 (P2-P3).]]<br />
<br />
[[Image:UNIPV10_19_10_10_MC42ABC_MC43ABC_MG42BC_MG43ABC_BLANK_MC1061_MG1655(TUTTI P3-P4).jpg|thumb|300px|center|The order of the samples was the follow: MC42(A,B,C), MC43(A,B,C), MG42(B,C), MG43(A,B,C), blank, MC1061, MG1655. Primers were BBa_K300977-BBa_K300978 (P3-P4).]]<br />
<br />
<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 20th==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 21st==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 22nd==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 23rd==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
<br><br><br />
<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana2| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana4| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana3Team:UNIPV-Pavia/Calendar/October/settimana32010-10-25T10:00:31Z<p>Matteo: /* October, 19th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 3</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 18th==<br />
<br />
We have finally completed the parts transfer to <partinfo>pSB1C3</partinfo> plasmid.<br />
All the parts that will be sent to the registry were previously screened by UNIPV-Pavia TEAM, as reported in this notebook and all of them were sequenced and sequencing results were correct.<br />
<br />
After parts have been moved to the shipping plasmid and checked by agarose gel electrophoresis.<br />
<br />
The parts we sent to MIT and submitted to the registry are:<br />
<br />
<table border='1' align='center'><br />
<tr><th>Part name</th><th>Wiki name</th><th>Plasmid</th><th>Number</th></tr><br />
<tr><td><partinfo>BBa_K300005</partinfo></td><td>I0</td><td>pSB1C3</td><td>1</td></tr><br />
<tr><td><partinfo>BBa_K300006</partinfo></td><td>I1</td><td>pSB1C3</td><td>2</td></tr><br />
<tr><td><partinfo>BBa_K300009</partinfo></td><td>I3</td><td>pSB1C3</td><td>3</td></tr><br />
<tr><td><partinfo>BBa_K300008</partinfo></td><td>I5</td><td>pSB1C3</td><td>4</td></tr><br />
<tr><td><partinfo>BBa_K300024</partinfo></td><td>I7</td><td>pSB1C3</td><td>5</td></tr><br />
<tr><td><partinfo>BBa_K300019</partinfo></td><td>I12</td><td>pSB1C3</td><td>6</td></tr><br />
<tr><td><partinfo>BBa_K300030</partinfo></td><td>I14</td><td>pSB1C3</td><td>7</td></tr><br />
<tr><td><partinfo>BBa_K300028</partinfo></td><td>I15</td><td>pSB1C3</td><td>8</td></tr><br />
<tr><td><partinfo>BBa_K300029</partinfo></td><td>I16</td><td>pSB1C3</td><td>9</td></tr><br />
<tr><td><partinfo>BBa_K300025</partinfo></td><td>I17</td><td>pSB1C3</td><td>10</td></tr><br />
<tr><td><partinfo>BBa_K300026</partinfo></td><td>I18</td><td>pSB1C3</td><td>11</td></tr><br />
<tr><td><partinfo>BBa_K300027</partinfo></td><td>I19</td><td>pSB1C3</td><td>12</td></tr><br />
<tr><td><partinfo>BBa_K300002</partinfo></td><td>I20</td><td>pSB1C3</td><td>13</td></tr><br />
<tr><td><partinfo>BBa_K300003</partinfo></td><td>I21</td><td>pSB1C3</td><td>14</td></tr><br />
<tr><td><partinfo>BBa_K300031</partinfo></td><td>I22</td><td>pSB1C3</td><td>15</td></tr><br />
<tr><td><partinfo>BBa_K300081</partinfo></td><td>I26</td><td>pSB1C3</td><td>16</td></tr><br />
<tr><td><partinfo>BBa_K300079</partinfo></td><td>I31</td><td>pSB1C3</td><td>17</td></tr><br />
<tr><td><partinfo>BBa_K300080</partinfo></td><td>I35</td><td>pSB1C3</td><td>18</td></tr><br />
<tr><td><partinfo>BBa_K300086</partinfo></td><td>I47</td><td>pSB1C3</td><td>19</td></tr><br />
<tr><td><partinfo>BBa_K300088</partinfo></td><td>I48</td><td>pSB1C3</td><td>20</td></tr><br />
<tr><td><partinfo>BBa_K300090</partinfo></td><td>I49</td><td>pSB1C3</td><td>21</td></tr><br />
<tr><td><partinfo>BBa_K300082</partinfo></td><td>I55</td><td>pSB1C3</td><td>22</td></tr><br />
<tr><td><partinfo>BBa_K300084</partinfo></td><td>I78</td><td>pSB1C3</td><td>23</td></tr><br />
<tr><td><partinfo>BBa_K300083</partinfo></td><td>I79</td><td>pSB1C3</td><td>24</td></tr><br />
<tr><td><partinfo>BBa_K300010</partinfo></td><td>I80</td><td>pSB1C3</td><td>25</td></tr><br />
<tr><td><partinfo>BBa_K300007</partinfo></td><td>I70 - BioBrick</td><td><partinfo>BBa_K300001</partinfo></td><td>26</td></tr><br />
<tr><td><partinfo>BBa_K300007</partinfo></td><td>I70 - vector</td><td><partinfo>BBa_K300001</partinfo></td><td>27</td></tr><br />
<tr><td><partinfo>BBa_K300004</partinfo></td><td>INTEIN</td><td>pSB1C3</td><td>28</td></tr><br />
<tr><td><partinfo>BBa_I52002</partinfo></td><td>I51</td><td><partinfo>BBa_K300000</partinfo></td><td>29</td></tr><br />
</table><br />
<br />
<br />
The parts we have in freezer and we only submitted to the Registry are:<br />
<table border='1' align='center'><br />
<tr><th>Part name</th><th>Wiki name</th><th>Plasmid</th></tr><br />
<tr><td><partinfo>BBa_K300021</partinfo></td><td>I8</td><td><partinfo>BBa_J61002</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300022</partinfo></td><td>I9</td><td><partinfo>BBa_J61002</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300023</partinfo></td><td>I10</td><td><partinfo>BBa_J61002</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300091</partinfo></td><td>I63</td><td><partinfo>pSB1AK3</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300092</partinfo></td><td>I64</td><td><partinfo>pSB1A2</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300099</partinfo></td><td>I65</td><td><partinfo>pSB1A2</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300980</partinfo></td><td>YEAST MR.G</td><td>pMA commercial vector</td></tr><br />
<tr><td><partinfo>BBa_K300982</partinfo></td><td>CREAM</td><td>-</td></tr><br />
<tr><td><partinfo>BBa_K300983</partinfo></td><td>CRIM MR.G</td><td>pMK-RQ commercial vector</td></tr><br />
<!--tr><td><partinfo></partinfo></td><td></td><td><partinfo></partinfo></td></tr--><br />
</table><br />
<br />
<br />
----<br />
In order to obtain better results with screening PCR for the E. coli genome integration project, we perform amplifications using an annealing temperature of 63°C.<br />
Screening PCR was performed on MC42(A-B-C), MC43(A-B-C), MG42(B-C), MG43(A-B-C), MC1061 and MG1655. Primers used were BBa_K300975-BBa_K300976, BBa_K300976-BBa_K300977, BBa_K300977-BBa_K300978.<br />
<br />
[[Image:UNIPV10_19-10-10_MC42ABC_MC43ABC_MG42BC_MG43ABC_BLANK_MC1061_MG1655(TUTTO_P1_P2).jpg|thumb|300px|center|The order of the samples was the follow: MC42(A,B,C), MC43(A,B,C), MG42(B,C), MG43(A,B,C), blank, MC1061, MG1655. Primers were BBa_K300975-BBa_K300976.]]<br />
<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 19th==<br />
<b>Wiki update and definition of results.</b><br />
<br />
----<br />
Every clones of E. coli genome integration project were amplified with PCR to verify their lengths and their positions into the genome.<br />
<br />
[[Image:UNIPV10_19_10_10_MC42ABC_MC43ABC_MG42BC_MG43ABC_BLANK_MC1061_MG1655(TUTTI P1-P2).jpg|thumb|300px|center|The order of the samples was the follow: MC42(A,B,C), MC43(A,B,C), MG42(B,C), MG43(A,B,C), blank, MC1061, MG1655. Primers were BBa_K300975-BBa_K300976 (P1-P2).]]<br />
<br />
[[Image:UNIPV10_19_10_10_MC42ABC_MC43ABC_MG42BC_MG43ABC_BLANK_MC1061_MG1655(TUTTI P2-P3).jpg|thumb|300px|center|The order of the samples was the follow: MC42(A,B,C), MC43(A,B,C), MG42(B,C), MG43(A,B,C), blank, MC1061, MG1655. Primers were BBa_K300976-BBa_K300977 (P2-P3).]]<br />
<br />
[[Image:UNIPV10_19_10_10_MC42ABC_MC43ABC_MG42BC_MG43ABC_BLANK_MC1061_MG1655(TUTTI P3-P4).jpg|thumb|300px|center|The order of the samples was the follow: MC42(A,B,C), MC43(A,B,C), MG42(B,C), MG43(A,B,C), blank, MC1061, MG1655. Primers were BBa_K300977-BBa_K300978 (P3-P4).]]<br />
<br />
<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 20th==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 21st==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 22nd==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 23rd==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
<br><br><br />
<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana2| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana4| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana3Team:UNIPV-Pavia/Calendar/October/settimana32010-10-25T09:53:16Z<p>Matteo: /* October, 19th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 3</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 18th==<br />
<br />
We have finally completed the parts transfer to <partinfo>pSB1C3</partinfo> plasmid.<br />
All the parts that will be sent to the registry were previously screened by UNIPV-Pavia TEAM, as reported in this notebook and all of them were sequenced and sequencing results were correct.<br />
<br />
After parts have been moved to the shipping plasmid and checked by agarose gel electrophoresis.<br />
<br />
The parts we sent to MIT and submitted to the registry are:<br />
<br />
<table border='1' align='center'><br />
<tr><th>Part name</th><th>Wiki name</th><th>Plasmid</th><th>Number</th></tr><br />
<tr><td><partinfo>BBa_K300005</partinfo></td><td>I0</td><td>pSB1C3</td><td>1</td></tr><br />
<tr><td><partinfo>BBa_K300006</partinfo></td><td>I1</td><td>pSB1C3</td><td>2</td></tr><br />
<tr><td><partinfo>BBa_K300009</partinfo></td><td>I3</td><td>pSB1C3</td><td>3</td></tr><br />
<tr><td><partinfo>BBa_K300008</partinfo></td><td>I5</td><td>pSB1C3</td><td>4</td></tr><br />
<tr><td><partinfo>BBa_K300024</partinfo></td><td>I7</td><td>pSB1C3</td><td>5</td></tr><br />
<tr><td><partinfo>BBa_K300019</partinfo></td><td>I12</td><td>pSB1C3</td><td>6</td></tr><br />
<tr><td><partinfo>BBa_K300030</partinfo></td><td>I14</td><td>pSB1C3</td><td>7</td></tr><br />
<tr><td><partinfo>BBa_K300028</partinfo></td><td>I15</td><td>pSB1C3</td><td>8</td></tr><br />
<tr><td><partinfo>BBa_K300029</partinfo></td><td>I16</td><td>pSB1C3</td><td>9</td></tr><br />
<tr><td><partinfo>BBa_K300025</partinfo></td><td>I17</td><td>pSB1C3</td><td>10</td></tr><br />
<tr><td><partinfo>BBa_K300026</partinfo></td><td>I18</td><td>pSB1C3</td><td>11</td></tr><br />
<tr><td><partinfo>BBa_K300027</partinfo></td><td>I19</td><td>pSB1C3</td><td>12</td></tr><br />
<tr><td><partinfo>BBa_K300002</partinfo></td><td>I20</td><td>pSB1C3</td><td>13</td></tr><br />
<tr><td><partinfo>BBa_K300003</partinfo></td><td>I21</td><td>pSB1C3</td><td>14</td></tr><br />
<tr><td><partinfo>BBa_K300031</partinfo></td><td>I22</td><td>pSB1C3</td><td>15</td></tr><br />
<tr><td><partinfo>BBa_K300081</partinfo></td><td>I26</td><td>pSB1C3</td><td>16</td></tr><br />
<tr><td><partinfo>BBa_K300079</partinfo></td><td>I31</td><td>pSB1C3</td><td>17</td></tr><br />
<tr><td><partinfo>BBa_K300080</partinfo></td><td>I35</td><td>pSB1C3</td><td>18</td></tr><br />
<tr><td><partinfo>BBa_K300086</partinfo></td><td>I47</td><td>pSB1C3</td><td>19</td></tr><br />
<tr><td><partinfo>BBa_K300088</partinfo></td><td>I48</td><td>pSB1C3</td><td>20</td></tr><br />
<tr><td><partinfo>BBa_K300090</partinfo></td><td>I49</td><td>pSB1C3</td><td>21</td></tr><br />
<tr><td><partinfo>BBa_K300082</partinfo></td><td>I55</td><td>pSB1C3</td><td>22</td></tr><br />
<tr><td><partinfo>BBa_K300084</partinfo></td><td>I78</td><td>pSB1C3</td><td>23</td></tr><br />
<tr><td><partinfo>BBa_K300083</partinfo></td><td>I79</td><td>pSB1C3</td><td>24</td></tr><br />
<tr><td><partinfo>BBa_K300010</partinfo></td><td>I80</td><td>pSB1C3</td><td>25</td></tr><br />
<tr><td><partinfo>BBa_K300007</partinfo></td><td>I70 - BioBrick</td><td><partinfo>BBa_K300001</partinfo></td><td>26</td></tr><br />
<tr><td><partinfo>BBa_K300007</partinfo></td><td>I70 - vector</td><td><partinfo>BBa_K300001</partinfo></td><td>27</td></tr><br />
<tr><td><partinfo>BBa_K300004</partinfo></td><td>INTEIN</td><td>pSB1C3</td><td>28</td></tr><br />
<tr><td><partinfo>BBa_I52002</partinfo></td><td>I51</td><td><partinfo>BBa_K300000</partinfo></td><td>29</td></tr><br />
</table><br />
<br />
<br />
The parts we have in freezer and we only submitted to the Registry are:<br />
<table border='1' align='center'><br />
<tr><th>Part name</th><th>Wiki name</th><th>Plasmid</th></tr><br />
<tr><td><partinfo>BBa_K300021</partinfo></td><td>I8</td><td><partinfo>BBa_J61002</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300022</partinfo></td><td>I9</td><td><partinfo>BBa_J61002</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300023</partinfo></td><td>I10</td><td><partinfo>BBa_J61002</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300091</partinfo></td><td>I63</td><td><partinfo>pSB1AK3</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300092</partinfo></td><td>I64</td><td><partinfo>pSB1A2</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300099</partinfo></td><td>I65</td><td><partinfo>pSB1A2</partinfo></td></tr><br />
<tr><td><partinfo>BBa_K300980</partinfo></td><td>YEAST MR.G</td><td>pMA commercial vector</td></tr><br />
<tr><td><partinfo>BBa_K300982</partinfo></td><td>CREAM</td><td>-</td></tr><br />
<tr><td><partinfo>BBa_K300983</partinfo></td><td>CRIM MR.G</td><td>pMK-RQ commercial vector</td></tr><br />
<!--tr><td><partinfo></partinfo></td><td></td><td><partinfo></partinfo></td></tr--><br />
</table><br />
<br />
<br />
----<br />
In order to obtain better results with screening PCR for the E. coli genome integration project, we perform amplifications using an annealing temperature of 63°C.<br />
Screening PCR was performed on MC42(A-B-C), MC43(A-B-C), MG42(B-C), MG43(A-B-C), MC1061 and MG1655. Primers used were BBa_K300975-BBa_K300976, BBa_K300976-BBa_K300977, BBa_K300977-BBa_K300978.<br />
<br />
[[Image:UNIPV10_19-10-10_MC42ABC_MC43ABC_MG42BC_MG43ABC_BLANK_MC1061_MG1655(TUTTO_P1_P2).jpg|thumb|300px|center|The order of the samples was the follow: MC42(A,B,C), MC43(A,B,C), MG42(B,C), MG43(A,B,C), blank, MC1061, MG1655. Primers were BBa_K300975-BBa_K300976.]]<br />
<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 19th==<br />
<b>Wiki update and definition of results.</b><br />
<br />
----<br />
Every clones of E. coli genome integration project were amplified with PCR to verify their lengths and their positions.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 20th==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 21st==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 22nd==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 23rd==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
<br><br><br />
<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana2| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana4| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/File:UNIPV10_19-10-10_MC42ABC_MC43ABC_MG42BC_MG43ABC_BLANK_MC1061_MG1655(TUTTO_P1_P2).jpgFile:UNIPV10 19-10-10 MC42ABC MC43ABC MG42BC MG43ABC BLANK MC1061 MG1655(TUTTO P1 P2).jpg2010-10-22T15:47:02Z<p>Matteo: </p>
<hr />
<div></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana3Team:UNIPV-Pavia/Calendar/October/settimana32010-10-22T15:46:25Z<p>Matteo: /* October, 18th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 3</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 18th==<br />
<br />
We have finally completed the parts transfer to <partinfo>pSB1C3</partinfo> plasmid.<br />
All the parts that will be sent to the registry were previously screened by UNIPV-Pavia TEAM, as reported in this notebook and all of them were sequenced and sequencing results were correct.<br />
<br />
After parts have been moved to the shipping plasmid and checked by agarose gel electrophoresis.<br />
<br />
The parts we sent to MIT are:<br />
<br />
<table border='1' align='center'><br />
<tr><th>Part name</th><th>Wiki name</th><th>Plasmid</th><th>Number</th></tr><br />
<tr><td><partinfo>BBa_K300005</partinfo></td><td>I0</td><td>pSB1C3</td><td>1</td></tr><br />
<tr><td><partinfo>BBa_K300006</partinfo></td><td>I1</td><td>pSB1C3</td><td>2</td></tr><br />
<tr><td><partinfo>BBa_K300009</partinfo></td><td>I3</td><td>pSB1C3</td><td>3</td></tr><br />
<tr><td><partinfo>BBa_K300008</partinfo></td><td>I5</td><td>pSB1C3</td><td>4</td></tr><br />
<tr><td><partinfo>BBa_K300024</partinfo></td><td>I7</td><td>pSB1C3</td><td>5</td></tr><br />
<tr><td><partinfo>BBa_K300019</partinfo></td><td>I12</td><td>pSB1C3</td><td>6</td></tr><br />
<tr><td><partinfo>BBa_K300030</partinfo></td><td>I14</td><td>pSB1C3</td><td>7</td></tr><br />
<tr><td><partinfo>BBa_K300028</partinfo></td><td>I15</td><td>pSB1C3</td><td>8</td></tr><br />
<tr><td><partinfo>BBa_K300029</partinfo></td><td>I16</td><td>pSB1C3</td><td>9</td></tr><br />
<tr><td><partinfo>BBa_K300025</partinfo></td><td>I17</td><td>pSB1C3</td><td>10</td></tr><br />
<tr><td><partinfo>BBa_K300026</partinfo></td><td>I18</td><td>pSB1C3</td><td>11</td></tr><br />
<tr><td><partinfo>BBa_K300027</partinfo></td><td>I19</td><td>pSB1C3</td><td>12</td></tr><br />
<tr><td><partinfo>BBa_K300002</partinfo></td><td>I20</td><td>pSB1C3</td><td>13</td></tr><br />
<tr><td><partinfo>BBa_K300003</partinfo></td><td>I21</td><td>pSB1C3</td><td>14</td></tr><br />
<tr><td><partinfo>BBa_K300031</partinfo></td><td>I22</td><td>pSB1C3</td><td>15</td></tr><br />
<tr><td><partinfo>BBa_K300081</partinfo></td><td>I26</td><td>pSB1C3</td><td>16</td></tr><br />
<tr><td><partinfo>BBa_K300079</partinfo></td><td>I31</td><td>pSB1C3</td><td>17</td></tr><br />
<tr><td><partinfo>BBa_K300080</partinfo></td><td>I35</td><td>pSB1C3</td><td>18</td></tr><br />
<tr><td><partinfo>BBa_K300086</partinfo></td><td>I47</td><td>pSB1C3</td><td>19</td></tr><br />
<tr><td><partinfo>BBa_K300088</partinfo></td><td>I48</td><td>pSB1C3</td><td>20</td></tr><br />
<tr><td><partinfo>BBa_K300090</partinfo></td><td>I49</td><td>pSB1C3</td><td>21</td></tr><br />
<tr><td><partinfo>BBa_K300082</partinfo></td><td>I55</td><td>pSB1C3</td><td>22</td></tr><br />
<tr><td><partinfo>BBa_K300084</partinfo></td><td>I78</td><td>pSB1C3</td><td>23</td></tr><br />
<tr><td><partinfo>BBa_K300083</partinfo></td><td>I79</td><td>pSB1C3</td><td>24</td></tr><br />
<tr><td><partinfo>BBa_K300010</partinfo></td><td>I80</td><td>pSB1C3</td><td>25</td></tr><br />
<tr><td><partinfo>BBa_K300007</partinfo></td><td>I70 - BioBrick</td><td><partinfo>BBa_K300001</partinfo></td><td>26</td></tr><br />
<tr><td><partinfo>BBa_K300007</partinfo></td><td>I70 - vector</td><td><partinfo>BBa_K300001</partinfo></td><td>27</td></tr><br />
<tr><td><partinfo>BBa_K300004</partinfo></td><td>INTEIN</td><td>pSB1C3</td><td>28</td></tr><br />
<tr><td><partinfo>BBa_I52002</partinfo></td><td>I51</td><td><partinfo>BBa_K300000</partinfo></td><td>29</td></tr><br />
<br />
</table><br />
<br />
<br />
----<br />
In order to obtain better results with screening PCR for the E. coli genome integration project, we perform amplifications using an annealing temperature of 63°C.<br />
Screening PCR was performed on MC42(A-B-C), MC43(A-B-C), MG42(B-C), MG43(A-B-C), MC1061 and MG1655. Primers used were BBa_K300975-BBa_K300976, BBa_K300976-BBa_K300977, BBa_K300977-BBa_K300978.<br />
<br />
[[Image:UNIPV10_19-10-10_MC42ABC_MC43ABC_MG42BC_MG43ABC_BLANK_MC1061_MG1655(TUTTO_P1_P2).jpg|thumb|300px|center|The order of the samples was the follow: MC42(A,B,C), MC43(A,B,C), MG42(B,C), MG42(A,B,C), blank, MC1061, MG1655. Primers were BBa_K300975-BBa_K300976.]]<br />
<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 19th==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 20th==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 21st==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 22nd==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 23rd==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
<br><br><br />
<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana2| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana4| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana3Team:UNIPV-Pavia/Calendar/October/settimana32010-10-22T15:42:57Z<p>Matteo: /* October, 18th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 3</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 18th==<br />
<br />
We have finally completed the parts transfer to <partinfo>pSB1C3</partinfo> plasmid.<br />
All the parts that will be sent to the registry were previously screened by UNIPV-Pavia TEAM, as reported in this notebook and all of them were sequenced and sequencing results were correct.<br />
<br />
After parts have been moved to the shipping plasmid and checked by agarose gel electrophoresis.<br />
<br />
The parts we sent to MIT are:<br />
<br />
<table border='1' align='center'><br />
<tr><th>Part name</th><th>Wiki name</th><th>Plasmid</th><th>Number</th></tr><br />
<tr><td><partinfo>BBa_K300005</partinfo></td><td>I0</td><td>pSB1C3</td><td>1</td></tr><br />
<tr><td><partinfo>BBa_K300006</partinfo></td><td>I1</td><td>pSB1C3</td><td>2</td></tr><br />
<tr><td><partinfo>BBa_K300009</partinfo></td><td>I3</td><td>pSB1C3</td><td>3</td></tr><br />
<tr><td><partinfo>BBa_K300008</partinfo></td><td>I5</td><td>pSB1C3</td><td>4</td></tr><br />
<tr><td><partinfo>BBa_K300024</partinfo></td><td>I7</td><td>pSB1C3</td><td>5</td></tr><br />
<tr><td><partinfo>BBa_K300019</partinfo></td><td>I12</td><td>pSB1C3</td><td>6</td></tr><br />
<tr><td><partinfo>BBa_K300030</partinfo></td><td>I14</td><td>pSB1C3</td><td>7</td></tr><br />
<tr><td><partinfo>BBa_K300028</partinfo></td><td>I15</td><td>pSB1C3</td><td>8</td></tr><br />
<tr><td><partinfo>BBa_K300029</partinfo></td><td>I16</td><td>pSB1C3</td><td>9</td></tr><br />
<tr><td><partinfo>BBa_K300025</partinfo></td><td>I17</td><td>pSB1C3</td><td>10</td></tr><br />
<tr><td><partinfo>BBa_K300026</partinfo></td><td>I18</td><td>pSB1C3</td><td>11</td></tr><br />
<tr><td><partinfo>BBa_K300027</partinfo></td><td>I19</td><td>pSB1C3</td><td>12</td></tr><br />
<tr><td><partinfo>BBa_K300002</partinfo></td><td>I20</td><td>pSB1C3</td><td>13</td></tr><br />
<tr><td><partinfo>BBa_K300003</partinfo></td><td>I21</td><td>pSB1C3</td><td>14</td></tr><br />
<tr><td><partinfo>BBa_K300031</partinfo></td><td>I22</td><td>pSB1C3</td><td>15</td></tr><br />
<tr><td><partinfo>BBa_K300081</partinfo></td><td>I26</td><td>pSB1C3</td><td>16</td></tr><br />
<tr><td><partinfo>BBa_K300079</partinfo></td><td>I31</td><td>pSB1C3</td><td>17</td></tr><br />
<tr><td><partinfo>BBa_K300080</partinfo></td><td>I35</td><td>pSB1C3</td><td>18</td></tr><br />
<tr><td><partinfo>BBa_K300086</partinfo></td><td>I47</td><td>pSB1C3</td><td>19</td></tr><br />
<tr><td><partinfo>BBa_K300088</partinfo></td><td>I48</td><td>pSB1C3</td><td>20</td></tr><br />
<tr><td><partinfo>BBa_K300090</partinfo></td><td>I49</td><td>pSB1C3</td><td>21</td></tr><br />
<tr><td><partinfo>BBa_K300082</partinfo></td><td>I55</td><td>pSB1C3</td><td>22</td></tr><br />
<tr><td><partinfo>BBa_K300084</partinfo></td><td>I78</td><td>pSB1C3</td><td>23</td></tr><br />
<tr><td><partinfo>BBa_K300083</partinfo></td><td>I79</td><td>pSB1C3</td><td>24</td></tr><br />
<tr><td><partinfo>BBa_K300010</partinfo></td><td>I80</td><td>pSB1C3</td><td>25</td></tr><br />
<tr><td><partinfo>BBa_K300007</partinfo></td><td>I70 - BioBrick</td><td><partinfo>BBa_K300001</partinfo></td><td>26</td></tr><br />
<tr><td><partinfo>BBa_K300007</partinfo></td><td>I70 - vector</td><td><partinfo>BBa_K300001</partinfo></td><td>27</td></tr><br />
<tr><td><partinfo>BBa_K300004</partinfo></td><td>INTEIN</td><td>pSB1C3</td><td>28</td></tr><br />
<tr><td><partinfo>BBa_I52002</partinfo></td><td>I51</td><td><partinfo>BBa_K300000</partinfo></td><td>29</td></tr><br />
<br />
</table><br />
<br />
<br />
----<br />
In order to obtain better results with screening PCR for the E. coli genome integration project, we perform amplifications using an annealing temperature of 63°C.<br />
Screening PCR was performed on MC42(A-B-C), MC43(A-B-C), MG42(B-C), MG43(A-B-C), MC1061 and MG1655. Primers used were BBa_K300975-BBa_K300976, BBa_K300976-BBa_K300977, BBa_K300977-BBa_K300978.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 19th==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 20th==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 21st==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 22nd==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 23rd==<br />
<b>Wiki update and definition of results.</b><br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
<br><br><br />
<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana2| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana4| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana2Team:UNIPV-Pavia/Calendar/October/settimana22010-10-22T15:37:20Z<p>Matteo: /* October, 13th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 2</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 11th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
Inoculum of MC42-A, MC43-A, MG42-B, MG43-A, MC43_flip-a,b,c, MG43_flip-a,b,c, MG1655.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 12th==<br />
<br />
Screening PCR was performed on MC42, MG42, MC43, MG43 and MG1655 with the following primers:<br />
<br />
-BBa_K300975, BBa_K300976 (P1-P2)<br />
<br />
-BBa_K300976, BBa_K300977 (P2-P3)<br />
<br />
-BBa_K300977, BBa_K300978 (P3-P4).<br />
<br />
[[Image:UNIPV10_12_10_2010_TOUCH_DOWN_PCR_CREAM.jpg|thumb|300px|center|The order of the samples were the follow: MC42, MC43, MG42, MG43, Cneg (MG1655), blank. Primers were (from left to right) BBa_K300975-BBa_K300976, BBa_K300976-BBa_K300977, BBa_K300977-BBa_K300978.]]<br />
<br />
Screening PCR was performed on MC43_flip(A,B,C), MG43_flip(A,B,C) and MG1655 with the following primers:<br />
<br />
-BBa_K300975, BBa_K300978 (P1-P4)<br />
<br />
-BBa_G00100,BBa_G00101 (VF2-VR)<br />
<br />
-BBa_K300977, BBa_K300978 (P3-P4).<br />
<br />
[[Image:UNIPV1013_10_10_mcflippati(p1p4)_mgflippati(p1p4)_mcflippati(p3p4) _mgflippati(p3p4).jpg|thumb|300px|center|The order of the samples was the follow: MC43_flip(A,B,C), MG43_flip(A,B,C), Cneg (MG1655), blank. Primers were (from left to right) BBa_K300975-BBa_K300978, BBa_K300977-BBa_K300978.]]<br />
<br />
[[Image:UNIPV10_13-10-10_mcflippati(vf2-vr)_mgflippati(vf2-vr)_cneg_blank.jpg|thumb|300px|center|The order of the samples was the follow: MC43_flip(A,B,C), MG43_flip(A,B,C), Cneg (MG1655), blank. Primers were (from left to right) BBa_G00100-BBa_G00101.]]<br />
<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 13th==<br />
For unknown reasons MC43_C didn't show any bands so we decided to perform again the screening with the same primers. At the same time we performed the screening PCR on the two negative controls with the primers BBa_K300977-BBa_K300978 and BBa_G00100-BBa_G00101. The results were the follows:<br />
<br />
[[Image:UNIPV10_14_10_10_PCR_Cneg(p3p4)_blank_MC43Cflip(LB-M9)(p1p4)_blank_Cneg(vf2vr)_blank.jpg|thumb|300px|center|The order of the samples was the follow: MG1655 (from 2 to 5) with BBa_K300977-BBa_K300978 primers, blank, MC43_flipC-1, MC43_flipC-2 with primers BBa_K300975-BBa_K300978, blank and MC1061 (from 10 to 13) with primers BBa_G00100-BBa_G00101]]<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 14th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 15th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 16th==<br />
<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
<br><br><br />
<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana1| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana3| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana2Team:UNIPV-Pavia/Calendar/October/settimana22010-10-22T15:37:00Z<p>Matteo: /* October, 12th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 2</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 11th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
Inoculum of MC42-A, MC43-A, MG42-B, MG43-A, MC43_flip-a,b,c, MG43_flip-a,b,c, MG1655.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 12th==<br />
<br />
Screening PCR was performed on MC42, MG42, MC43, MG43 and MG1655 with the following primers:<br />
<br />
-BBa_K300975, BBa_K300976 (P1-P2)<br />
<br />
-BBa_K300976, BBa_K300977 (P2-P3)<br />
<br />
-BBa_K300977, BBa_K300978 (P3-P4).<br />
<br />
[[Image:UNIPV10_12_10_2010_TOUCH_DOWN_PCR_CREAM.jpg|thumb|300px|center|The order of the samples were the follow: MC42, MC43, MG42, MG43, Cneg (MG1655), blank. Primers were (from left to right) BBa_K300975-BBa_K300976, BBa_K300976-BBa_K300977, BBa_K300977-BBa_K300978.]]<br />
<br />
Screening PCR was performed on MC43_flip(A,B,C), MG43_flip(A,B,C) and MG1655 with the following primers:<br />
<br />
-BBa_K300975, BBa_K300978 (P1-P4)<br />
<br />
-BBa_G00100,BBa_G00101 (VF2-VR)<br />
<br />
-BBa_K300977, BBa_K300978 (P3-P4).<br />
<br />
[[Image:UNIPV1013_10_10_mcflippati(p1p4)_mgflippati(p1p4)_mcflippati(p3p4) _mgflippati(p3p4).jpg|thumb|300px|center|The order of the samples was the follow: MC43_flip(A,B,C), MG43_flip(A,B,C), Cneg (MG1655), blank. Primers were (from left to right) BBa_K300975-BBa_K300978, BBa_K300977-BBa_K300978.]]<br />
<br />
[[Image:UNIPV10_13-10-10_mcflippati(vf2-vr)_mgflippati(vf2-vr)_cneg_blank.jpg|thumb|300px|center|The order of the samples was the follow: MC43_flip(A,B,C), MG43_flip(A,B,C), Cneg (MG1655), blank. Primers were (from left to right) BBa_G00100-BBa_G00101.]]<br />
<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 13th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 14th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 15th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 16th==<br />
<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
<br><br><br />
<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana1| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana3| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/File:UNIPV10_14_10_10_PCR_Cneg(p3p4)_blank_MC43Cflip(LB-M9)(p1p4)_blank_Cneg(vf2vr)_blank.jpgFile:UNIPV10 14 10 10 PCR Cneg(p3p4) blank MC43Cflip(LB-M9)(p1p4) blank Cneg(vf2vr) blank.jpg2010-10-22T15:21:04Z<p>Matteo: </p>
<hr />
<div></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana2Team:UNIPV-Pavia/Calendar/October/settimana22010-10-22T15:20:13Z<p>Matteo: /* October, 12th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 2</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 11th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
Inoculum of MC42-A, MC43-A, MG42-B, MG43-A, MC43_flip-a,b,c, MG43_flip-a,b,c, MG1655.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 12th==<br />
<br />
Screening PCR was performed on MC42, MG42, MC43, MG43 and MG1655 with the following primers:<br />
<br />
-BBa_K300975, BBa_K300976 (P1-P2)<br />
<br />
-BBa_K300976, BBa_K300977 (P2-P3)<br />
<br />
-BBa_K300977, BBa_K300978 (P3-P4).<br />
<br />
[[Image:UNIPV10_12_10_2010_TOUCH_DOWN_PCR_CREAM.jpg|thumb|300px|center|The order of the samples were the follow: MC42, MC43, MG42, MG43, Cneg (MG1655), blank. Primers were (from left to right) BBa_K300975-BBa_K300976, BBa_K300976-BBa_K300977, BBa_K300977-BBa_K300978.]]<br />
<br />
Screening PCR was performed on MC43_flip(A,B,C), MG43_flip(A,B,C) and MG1655 with the following primers:<br />
<br />
-BBa_K300975, BBa_K300978 (P1-P4)<br />
<br />
-BBa_G00100,BBa_G00101 (VF2-VR)<br />
<br />
-BBa_K300977, BBa_K300978 (P3-P4).<br />
<br />
[[Image:UNIPV1013_10_10_mcflippati(p1p4)_mgflippati(p1p4)_mcflippati(p3p4) _mgflippati(p3p4).jpg|thumb|300px|center|The order of the samples was the follow: MC43_flip(A,B,C), MG43_flip(A,B,C), Cneg (MG1655), blank. Primers were (from left to right) BBa_K300975-BBa_K300978, BBa_K300977-BBa_K300978.]]<br />
<br />
[[Image:UNIPV10_13-10-10_mcflippati(vf2-vr)_mgflippati(vf2-vr)_cneg_blank.jpg|thumb|300px|center|The order of the samples was the follow: MC43_flip(A,B,C), MG43_flip(A,B,C), Cneg (MG1655), blank. Primers were (from left to right) BBa_G00100-BBa_G00101.]]<br />
<br />
For unknown reasons MC43_C didn't show any bands so we decided to perform again the screening with the same primers. At the same time we performed the screening PCR on the two negative controls with the primers BBa_K300977-BBa_K300978 and BBa_G00100-BBa_G00101. The results were the follows:<br />
<br />
[[Image:UNIPV10_14_10_10_PCR_Cneg(p3p4)_blank_MC43Cflip(LB-M9)(p1p4)_blank_Cneg(vf2vr)_blank.jpg|thumb|300px|center|The order of the samples was the follow: MG1655 (from 2 to 5) with BBa_K300977-BBa_K300978 primers, blank, MC43_flipC-1, MC43_flipC-2 with primers BBa_K300975-BBa_K300978, blank and MC1061 (from 10 to 13) with primers BBa_G00100-BBa_G00101]]<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 13th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 14th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 15th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 16th==<br />
<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
<br><br><br />
<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana1| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana3| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana2Team:UNIPV-Pavia/Calendar/October/settimana22010-10-20T19:09:42Z<p>Matteo: /* October, 12th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 2</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 11th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
Inoculum of MC42-A, MC43-A, MG42-B, MG43-A, MC43_flip-a,b,c, MG43_flip-a,b,c, MG1655.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 12th==<br />
<br />
Screening PCR was performed on MC42, MG42, MC43, MG43 and MG1655 with the following primers:<br />
<br />
-BBa_K300975, BBa_K300976 (P1-P2)<br />
<br />
-BBa_K300976, BBa_K300977 (P2-P3)<br />
<br />
-BBa_K300977, BBa_K300978 (P3-P4).<br />
<br />
[[Image:UNIPV10_12_10_2010_TOUCH_DOWN_PCR_CREAM.jpg|thumb|300px|center|The order of the samples were the follow: MC42, MC43, MG42, MG43, Cneg (MG1655), blank. Primers were (from left to right) BBa_K300975-BBa_K300976, BBa_K300976-BBa_K300977, BBa_K300977-BBa_K300978.]]<br />
<br />
Screening PCR was performed on MC43_flip(A,B,C), MG43_flip(A,B,C) and MG1655 with the following primers:<br />
<br />
-BBa_K300975, BBa_K300978 (P1-P4)<br />
<br />
-BBa_G00100,BBa_G00101 (VF2-VR)<br />
<br />
-BBa_K300977, BBa_K300978 (P3-P4).<br />
<br />
[[Image:UNIPV1013_10_10_mcflippati(p1p4)_mgflippati(p1p4)_mcflippati(p3p4) _mgflippati(p3p4).jpg|thumb|300px|center|The order of the samples were the follow: MC43_flip(A,B,C), MG43_flip(A,B,C), Cneg (MG1655), blank. Primers were (from left to right) BBa_K300975-BBa_K300978, BBa_K300977-BBa_K300978.]]<br />
<br />
[[Image:UNIPV10_13-10-10_mcflippati(vf2-vr)_mgflippati(vf2-vr)_cneg_blank.jpg|thumb|300px|center|The order of the samples were the follow: MC43_flip(A,B,C), MG43_flip(A,B,C), Cneg (MG1655), blank. Primers were (from left to right) BBa_G00100-BBa_G00101.]]<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 13th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 14th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 15th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 16th==<br />
<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
<br><br><br />
<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana1| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana3| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana2Team:UNIPV-Pavia/Calendar/October/settimana22010-10-20T19:08:12Z<p>Matteo: /* October, 12th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 2</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 11th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
Inoculum of MC42-A, MC43-A, MG42-B, MG43-A, MC43_flip-a,b,c, MG43_flip-a,b,c, MG1655.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 12th==<br />
<br />
Screening PCR was performed on MC42, MG42, MC43, MG43 and MG1655 with the following primers:<br />
<br />
-BBa_K300975, BBa_K300976<br />
<br />
-BBa_K300976, BBa_K300977<br />
<br />
-BBa_K300977, BBa_K300978.<br />
<br />
[[Image:UNIPV10_12_10_2010_TOUCH_DOWN_PCR_CREAM.jpg|thumb|300px|center|The order of the samples were the follow: MC42, MC43, MG42, MG43, Cneg (MG1655), blank. Primers were (from left to right) BBa_K300975-BBa_K300976, BBa_K300976-BBa_K300977, BBa_K300977-BBa_K300978.]]<br />
<br />
Screening PCR was performed on MC43_flip(A,B,C), MG43_flip(A,B,C) and MG1655 with the following primers:<br />
<br />
-BBa_K300975, BBa_K300978<br />
<br />
-BBa_G00100,BBa_G00101<br />
<br />
-BBa_K300977, BBa_K300978.<br />
<br />
[[Image:UNIPV1013_10_10_mcflippati(p1p4)_mgflippati(p1p4)_mcflippati(p3p4) _mgflippati(p3p4).jpg|thumb|300px|center|The order of the samples were the follow: MC43_flip(A,B,C), MG43_flip(A,B,C), Cneg (MG1655), blank. Primers were (from left to right) BBa_K300975-BBa_K300978, BBa_K300977-BBa_K300978.]]<br />
<br />
[[Image:UNIPV10_13-10-10_mcflippati(vf2-vr)_mgflippati(vf2-vr)_cneg_blank.jpg|thumb|300px|center|The order of the samples were the follow: MC43_flip(A,B,C), MG43_flip(A,B,C), Cneg (MG1655), blank. Primers were (from left to right) BBa_G00100-BBa_G00101.]]<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 13th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 14th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 15th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 16th==<br />
<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
<br><br><br />
<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana1| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana3| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana2Team:UNIPV-Pavia/Calendar/October/settimana22010-10-20T18:41:14Z<p>Matteo: /* October, 11th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 2</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 11th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
Inoculum of MC42-A, MC43-A, MG42-B, MG43-A, MC43_flip-a,b,c, MG43_flip-a,b,c, MG1655.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 12th==<br />
<br />
Screening PCR was performed on MC42, MG42, MC43, MG43 and MG1655 with the following primers:<br />
<br />
-BBa_K300976, BBa_K300977<br />
<br />
-BBa_K300977, BBa_K300978<br />
<br />
-BBa_K300978, BBa_K300979.<br />
<br />
[[Image:UNIPV10_12_10_2010_TOUCH_DOWN_PCR_CREAM.jpg|thumb|300px|center|The order of the samples were the follow: MC42, MC43, MG42, MG43, Cneg (MG1655), blank. Primers were (from left to right) BBa_K300976-BBa_K300977, BBa_K300977-BBa_K300978, BBa_K300978-BBa_K300979.]]<br />
<br />
Screening PCR was performed on MC43_flip(A,B,C), MG43_flip(A,B,C) and MG1655 with the following primers:<br />
<br />
-BBa_K300976, BBa_K300979<br />
<br />
-BBa_G00100,BBa_G00101<br />
<br />
-BBa_K300978, BBa_K300979.<br />
<br />
[[Image:UNIPV1013_10_10_mcflippati(p1p4)_mgflippati(p1p4)_mcflippati(p3p4) _mgflippati(p3p4).jpg|thumb|300px|center|The order of the samples were the follow: MC43_flip(A,B,C), MG43_flip(A,B,C), Cneg (MG1655), blank. Primers were (from left to right) BBa_K300976-BBa_K300979, BBa_K300978-BBa_K300979.]]<br />
<br />
[[Image:UNIPV10_13-10-10_mcflippati(vf2-vr)_mgflippati(vf2-vr)_cneg_blank.jpg|thumb|300px|center|The order of the samples were the follow: MC43_flip(A,B,C), MG43_flip(A,B,C), Cneg (MG1655), blank. Primers were (from left to right) BBa_G00100-BBa_G00101.]]<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 13th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 14th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 15th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 16th==<br />
<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
<br><br><br />
<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana1| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana3| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/File:UNIPV10_13-10-10_mcflippati(vf2-vr)_mgflippati(vf2-vr)_cneg_blank.jpgFile:UNIPV10 13-10-10 mcflippati(vf2-vr) mgflippati(vf2-vr) cneg blank.jpg2010-10-20T18:36:20Z<p>Matteo: </p>
<hr />
<div></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana2Team:UNIPV-Pavia/Calendar/October/settimana22010-10-20T18:35:55Z<p>Matteo: /* October, 12th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 2</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 11th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
Inoculum of MC42-A, MC43-A, MG42-B, MG43-A, MC43_flip-a,b,c, MG43_flip-a,b,c, MC1061.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 12th==<br />
<br />
Screening PCR was performed on MC42, MG42, MC43, MG43 and MG1655 with the following primers:<br />
<br />
-BBa_K300976, BBa_K300977<br />
<br />
-BBa_K300977, BBa_K300978<br />
<br />
-BBa_K300978, BBa_K300979.<br />
<br />
[[Image:UNIPV10_12_10_2010_TOUCH_DOWN_PCR_CREAM.jpg|thumb|300px|center|The order of the samples were the follow: MC42, MC43, MG42, MG43, Cneg (MG1655), blank. Primers were (from left to right) BBa_K300976-BBa_K300977, BBa_K300977-BBa_K300978, BBa_K300978-BBa_K300979.]]<br />
<br />
Screening PCR was performed on MC43_flip(A,B,C), MG43_flip(A,B,C) and MG1655 with the following primers:<br />
<br />
-BBa_K300976, BBa_K300979<br />
<br />
-BBa_G00100,BBa_G00101<br />
<br />
-BBa_K300978, BBa_K300979.<br />
<br />
[[Image:UNIPV1013_10_10_mcflippati(p1p4)_mgflippati(p1p4)_mcflippati(p3p4) _mgflippati(p3p4).jpg|thumb|300px|center|The order of the samples were the follow: MC43_flip(A,B,C), MG43_flip(A,B,C), Cneg (MG1655), blank. Primers were (from left to right) BBa_K300976-BBa_K300979, BBa_K300978-BBa_K300979.]]<br />
<br />
[[Image:UNIPV10_13-10-10_mcflippati(vf2-vr)_mgflippati(vf2-vr)_cneg_blank.jpg|thumb|300px|center|The order of the samples were the follow: MC43_flip(A,B,C), MG43_flip(A,B,C), Cneg (MG1655), blank. Primers were (from left to right) BBa_G00100-BBa_G00101.]]<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 13th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 14th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 15th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 16th==<br />
<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
<br><br><br />
<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana1| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana3| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/File:UNIPV1013_10_10_mcflippati(p1p4)_mgflippati(p1p4)_mcflippati(p3p4)_mgflippati(p3p4).jpgFile:UNIPV1013 10 10 mcflippati(p1p4) mgflippati(p1p4) mcflippati(p3p4) mgflippati(p3p4).jpg2010-10-20T18:33:38Z<p>Matteo: </p>
<hr />
<div></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana2Team:UNIPV-Pavia/Calendar/October/settimana22010-10-20T18:33:15Z<p>Matteo: /* October, 12th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 2</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 11th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
Inoculum of MC42-A, MC43-A, MG42-B, MG43-A, MC43_flip-a,b,c, MG43_flip-a,b,c, MC1061.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 12th==<br />
<br />
Screening PCR was performed on MC42, MG42, MC43, MG43 and MG1655 with the following primers:<br />
<br />
-BBa_K300976, BBa_K300977<br />
<br />
-BBa_K300977, BBa_K300978<br />
<br />
-BBa_K300978, BBa_K300979.<br />
<br />
[[Image:UNIPV10_12_10_2010_TOUCH_DOWN_PCR_CREAM.jpg|thumb|300px|center|The order of the samples were the follow: MC42, MC43, MG42, MG43, Cneg (MG1655), blank. Primers were (from left to right) BBa_K300976-BBa_K300977, BBa_K300977-BBa_K300978, BBa_K300978-BBa_K300979.]]<br />
<br />
Screening PCR was performed on MC43_flip(A,B,C), MG43_flip(A,B,C) and MG1655 with the following primers:<br />
<br />
-BBa_K300976, BBa_K300979<br />
<br />
-BBa_G00100,BBa_G00101<br />
<br />
-BBa_K300978, BBa_K300979.<br />
<br />
[[Image:UNIPV1013_10_10_mcflippati(p1p4)_mgflippati(p1p4)_mcflippati(p3p4) _mgflippati(p3p4).jpg|thumb|300px|center|The order of the samples were the follow: MC43_flip(A,B,C), MG43_flip(A,B,C), Cneg (MG1655), blank. Primers were (from left to right) BBa_K300976-BBa_K300979, BBa_K300978-BBa_K300979.]]<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 13th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 14th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 15th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 16th==<br />
<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
<br><br><br />
<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana1| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana3| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana2Team:UNIPV-Pavia/Calendar/October/settimana22010-10-20T18:28:04Z<p>Matteo: /* October, 12th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 2</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 11th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
Inoculum of MC42-A, MC43-A, MG42-B, MG43-A, MC43_flip-a,b,c, MG43_flip-a,b,c, MC1061.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 12th==<br />
<br />
Screening PCR was performed on MC42, MG42, MC43, MG43 and MC1061 with the following primers:<br />
<br />
-BBa_K300976, BBa_K300977<br />
<br />
-BBa_K300977, BBa_K300978<br />
<br />
-BBa_K300978, BBa_K300979.<br />
<br />
[[Image:UNIPV10_12_10_2010_TOUCH_DOWN_PCR_CREAM.jpg|thumb|300px|center|The order of the samples were the follow: MC42, MC43, MG42, MG43, Cneg (MG1655), blank. Primers were (from left to right) BBa_K300976-BBa_K300977, BBa_K300977-BBa_K300978, BBa_K300978-BBa_K300979.]]<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 13th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 14th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 15th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 16th==<br />
<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
<br><br><br />
<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana1| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana3| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana2Team:UNIPV-Pavia/Calendar/October/settimana22010-10-20T18:27:21Z<p>Matteo: /* October, 12th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 2</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 11th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
Inoculum of MC42-A, MC43-A, MG42-B, MG43-A, MC43_flip-a,b,c, MG43_flip-a,b,c, MC1061.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 12th==<br />
<br />
Screening PCR was performed on MC42, MG42, MC43, MG43 and MC1061 with the following primers:<br />
<br />
-BBa_K300976, BBa_K300977<br />
<br />
-BBa_K300977, BBa_K300978<br />
<br />
-BBa_K300978, BBa_K300979.<br />
<br />
[[Image:UNIPV10_12_10_2010_TOUCH_DOWN_PCR_CREAM.jpg|thumb|200px|center|The order of the samples were the follow: MC42, MC43, MG42, MG43, Cneg (MG1655), blank. Primers were (from left to right) BBa_K300976-BBa_K300977, BBa_K300977-BBa_K300978, BBa_K300978-BBa_K300979.]]<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 13th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 14th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 15th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 16th==<br />
<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
<br><br><br />
<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana1| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana3| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/File:UNIPV10_12_10_2010_TOUCH_DOWN_PCR_CREAM.jpgFile:UNIPV10 12 10 2010 TOUCH DOWN PCR CREAM.jpg2010-10-20T18:24:20Z<p>Matteo: </p>
<hr />
<div></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana2Team:UNIPV-Pavia/Calendar/October/settimana22010-10-20T18:23:37Z<p>Matteo: /* October, 12th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 2</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 11th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
Inoculum of MC42-A, MC43-A, MG42-B, MG43-A, MC43_flip-a,b,c, MG43_flip-a,b,c, MC1061.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 12th==<br />
<br />
Screening PCR was performed on MC42, MG42, MC43, MG43 and MC1061 with the following primers:<br />
<br />
-BBa_K300976, BBa_K300977<br />
<br />
-BBa_K300977, BBa_K300978<br />
<br />
-BBa_K300978, BBa_K300979.<br />
<br />
[[Image:UNIPV10_12_10_2010_TOUCH_DOWN_PCR_CREAM.jpg|thumb|200px|center|The order of the samples were the follow: MC42, MC43, MG42, MG43, Cneg (MG1655), blank.]]<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 13th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 14th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 15th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 16th==<br />
<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
<br><br><br />
<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana1| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana3| Next week]]</td><br />
</tr><br />
</table><br />
<!-- fine table previous next week --><br />
</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana2Team:UNIPV-Pavia/Calendar/October/settimana22010-10-20T18:17:56Z<p>Matteo: /* October, 12th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 2</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 11th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
Inoculum of MC42-A, MC43-A, MG42-B, MG43-A, MC43_flip-a,b,c, MG43_flip-a,b,c, MC1061.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 12th==<br />
<br />
Screening PCR was performed on MC42, MG42, MC43, MG43 and MC1061 with the following primers:<br />
<br />
-BBa_K300976, BBa_K300977<br />
<br />
-BBa_K300977, BBa_K300978<br />
<br />
-BBa_K300978, BBa_K300979.<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 13th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 14th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 15th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 16th==<br />
<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
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<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/October/settimana1| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana3| Next week]]</td><br />
</tr><br />
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</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/September/settimana5Team:UNIPV-Pavia/Calendar/September/settimana52010-10-20T17:56:45Z<p>Matteo: /* October, 3rd */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana4|Week 4]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana5|Week 5]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>SEPTEMBER: WEEK 5</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==September, 27th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test27settembre|Tecan Test]]</font><br />
<br />
----<br />
<br />
MiniPrep and E-P digest for I62-a, I62-b, I62-c, I62-d, I62-e, I62-f<br />
<br />
DNA PCR for MyYeast 1-2-3<br />
<br />
Gel run<br />
[[Image:UNIPV10_27_09_2010_I62_screen.jpg|thumb|200px|center|I62 and MyYeast screening.]]<br />
<br />
<br />
I62-e was positive, so we made glycerol stock and stored it at -80°C.<br />
<br />
<br />
Protein electrophoresis for phasins.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 28th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test28settembre|Tecan Test]]</font><br />
<br />
----<br />
Miniprep and quantification for:<br />
*I1: 59,6 ng/ul<br />
*I4: 109,9 ng/ul<br />
*I53: 160,3 ng/ul<br />
*<partinfo>BBa_J04450</partinfo>-1:55,9 ng/ul<br />
*<partinfo>BBa_J04450</partinfo>-2: 47 ng/ul<br />
<br />
ON digestion of:<br />
*I1 (E-S)<br />
*I4 (E-S)<br />
*I53 (E-X)<br />
*<partinfo>BBa_J04450</partinfo>-1 (E-P)<br />
*<partinfo>BBa_J04450</partinfo>-2 (E-P)<br />
*MyYeast-3 (DNA already available) (E-S)<br />
*<partinfo>pSB4C5</partinfo> (DNA already available) (E-P)<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 29th==<br />
<br />
Colony PCR for 6 newly picked colonies.<br />
[[Image:UNIPV10_29_09_2010_I55_screen.jpg|thumb|200px|center|I55 colony PCR screening.]]<br />
No positives nor this time.<br />
----<br />
<br />
<br />
Gel run for all digested parts<br />
[[Image:UNIPV10_29_09_2010_digestions.jpg|thumb|200px|center|Digestions.]]<br />
and cut/gel purification for:<br />
*4C5 (E-P): 19,3 ng/ul<br />
*My Yeast 3 (E-S): 20,8 ng/ul<br />
*I1: 3,8 ng/ul<br />
*I4 (E-S): 14,2 ng/ul<br />
*I53 (E-X): 21,6 ng/ul<br />
*<partinfo>BBa_J04450</partinfo> (pLac-RBS-mRFP-TT) (E-P): 5,6 ng/ul<br />
*<partinfo>pSB1C3</partinfo>: 10 ng/ul<br />
<br />
<br />
Ligation of:<br />
{| align='center', border='1'<br />
| I70=MyYeast 3(E-S)+I4(E-S)<br />
|-<br />
| I71=MyYeast 3(E-S)+I1(E-S)<br />
|-<br />
|I72=<partinfo>BBa_J04450</partinfo>(E-P)+<partinfo>pSB4C5</partinfo><br />
|-<br />
|I73= I38 (E-S) + I53 (E-X)<br />
|}<br />
<br />
----<br />
<br />
Inoculum of MC43, MG43 in 5ml LB with chloramphenicol concentration of 12.5 ug/ml.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 30th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test16settembre|Tecan Test]]</font><br />
<br />
----<br />
<br />
Transformation of I73 (1ul) into ''E. coli'' TOP10. It was plated on LB+Amp 100 ug/ml agar plate.<br />
<br />
Trasformation of: <br />
*I70 (myYeast with I4, Ptef1-mOrange-tAdh1) in TOP10 and plated on LB+Amp<br />
*I71 (myYeast with I1, mOrange-tAdh1) in TOP10 and plated on LB+Amp<br />
*I72 (pLac-RFP in pSB4C5) in TOP10 and plated on LB+Cm 12,5<br />
<br />
All plates were incubated at 37°C ON<br />
<br />
<br />
Since I55 was negative again we decided to ligate it again (probably we had a bad digest for vector - I20 - and a good one for insert - we used I38 in other ligations without problems). We diegested already miniprepped DNA for three hours with 1 ul of enzymes:<br />
*I20: (E-X)<br />
<br />
Gel run/cut/extraction/quantification<br />
[[Image:UNIPV10_30_09_2010_I20_E_X.jpg|thumb|70px|center|I20 E-X digest.]]<br />
I20 E-X: 13,4 ng/ul<br />
<br />
We repeated I55 ligation using newly digested vector and already digested I38 (E-S)<br />
<br />
I55: I38 (E-S) + I20 (E-X)<br />
<br />
Inoculum into 5ml LB+Amp of:<br />
*I31<br />
*I35<br />
*I37<br />
*I57<br />
*I60<br />
*INTEIN<br />
*3x <partinfo>BBa_J04450</partinfo> (to take <partinfo>pSB1C3</partinfo>, so <partinfo>pSB1C3</partinfo> from now on)<br />
Cultures were let grow ON at 37°C, 220 rpm.<br />
<br />
----<br />
<br />
Inoculum of E. coli TOP10 in 1,5 ml LB, grown ON at 37°C 220 rpm. Tomorrow we will prepare competent cells!!<br />
<br />
Inoculum of:<br />
<br />
*S1 in 500ul M9+Cm 12,5<br />
*I47-S1 in 500ul M9+Amp+Cm 12,5<br />
*GFP-S1 in 500ul M9+Amp+Cm 12,5<br />
*RBS32 in 500ul M9+Amp<br />
<br />
Cultures were grown ON at 37°C 220 rpm.<br />
<br />
Tomorrow we will test the ability of these strains to produce GFP and to lyse when HSL is added.<br />
<br />
----<br />
In order to excise the chloramphenicol resistance cassette from the integrants in E.coli genome we transformed the cells with a plasmid (pCP20 plasmid) carrying the gene encoding for the flippase protein. This protein is able to cut fragment between two FRT flanking sequences. pCP20 was transformed in MC43-a and MG43-a and plated on LB+Amp agar plate.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 1st==<br />
<br />
This morning, we checked tranformed plates:<br />
<br />
*I72 showed NO colony!! :/ we will repeat this ligation next week<br />
*I70 and I71 showed colonies, 5 for each plate were picked and inoculated in 1ml LB+Amp to prepare glycerol stocks and for screening.<br />
*I73 showed colonies; six of them were picked and inoculated into 5 ml LB+Amp and let grow ON at 37°C, 220rpm in order to do E-P screeening the following day.<br />
<br />
----<br />
<br />
Transformation of I55 into ''E. coli'' DH5-alpha. It was plated on LB+Amp 100 agar plate and let grow ON at +37°C.<br />
<br />
----<br />
<br />
Miniprep and Nanodrop quantification for:<br />
*I31: 76,1 ng/ul<br />
*I35: 66,8 ng/ul<br />
*I60: 279,7 ng/ul<br />
(they were also sent sequencing)<br />
*INTEIN: 123,8 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-1: 104,8 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-2: 141,2 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-3: 113,5 ng/ul<br />
*I37: 174,9 ng/ul<br />
<br />
Digest:<br />
*<partinfo>pSB1C3</partinfo>: E-P<br />
*<partinfo>pSB1C3</partinfo>: E-S<br />
*<partinfo>pSB1C3</partinfo>: X-P<br />
*INTEIN: E-P<br />
*I60: E-S<br />
*I31: S-P<br />
*I35: E-P<br />
*I35: E-X<br />
*I37: E-X<br />
*I57: E-X<br />
<br />
Gel run/cut/extraction/quantification:<br />
{|align="center"<br />
|[[Image:UNIPV10_01_10_2010_digestioni_medio.jpg|thumb|200px|center|Intein, I31, I35, I37, I57, I60 digestions]] || [[Image:UNIPV10_01_10_2010_digestioni_psb1c3.jpg|thumb|150px|center|<partinfo>pSB1C3</partinfo> digested E-P (both vector and insert were kept), E-S, X-P.]]<br />
|}<br />
<br />
*I37 (E-X): 20,4 ng/ul<br />
*I57 (E-X): 17,6 ng/ul<br />
*I60 (E-S): 9,5 ng/ul<br />
They were stored at -20°C.<br />
*I31 (E-S): 5,4 ng/ul<br />
*I35 (E-P): 1,0 ng/ul ;-(<br />
*I35 (E-X): 14,4 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (E-P): 14,3 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (E-S): 15,2 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (X-P): 13,9 ng/ul<br />
<br />
ON ligation of:<br />
*I74: I31 (E-S) + I35 (E-X)<br />
*INTEIN_C3: INTEIN (E-P) + <partinfo>pSB1C3</partinfo> (E-P)<br />
<br />
----<br />
<br />
Single colony was picked from MC43 and MG43 plates and inoculated in 5 ml of LB and incubated at 37°C, 220 rpm ON.<br />
12 hours late 5 ul were streaked on non selective LB plates and incubated at 43°C ON in order to ensure the loss of pCP20 plasmid.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 2nd==<br />
<br />
PCR from colony for I55 (eleven colonies were picked).<br />
<br />
Gel run:<br />
<br />
[[Image:UNIPV10_02_10_2010_I55_screening.jpg|thumb|200px|center|I55 colony PCR screening.]]<br />
<br />
We decided to make glycerol stock for I55-1. Inoculum into 5 ml LB+Amp of the positive culture that was let grow ON at 37°C, 220 rpm.<br />
----<br />
Miniprep and quantification for I73-1/2/3/4/5/6:<br />
*I73-1: 154 ng/ul<br />
*I73-2: 177 ng/ul<br />
*I73-3: 154,7 ng/ul<br />
*I73-4: 145,5ng/ul<br />
*I73-5: 152,6 ng/ul<br />
*I73-6: 154,2 ng/ul<br />
1,5 hours E-P digest (for screening) and gel run<br />
<br />
[[Image:UNIPV10_02_10_2010_I73_screening.jpg|thumb|200px|center|I70, I71, I73, A9_4C5, A99_4C5 screening.]]<br />
<br />
As you can see they are (except for I73-6) all positive. So we stocked I73-1 and stored it at -80°C.<br />
<br />
----<br />
Transformation of I74 and INTEIN_C3 (1ul) in ''E. coli'' TOP10. They were plated on LB+Amp100 and LB+Cm34 agar plates respectively. They were let grow ON at +37°C.<br />
<br />
----<br />
Inoculum of I55-1 into 5 ml LB+Amp. ON growth at +37°C, 220 rpm.<br />
<br />
----<br />
<br />
3 colonies each plates were picked from MC43_flip and MG43_flip plates and inoculated in 5 ml of non selective LB. To confirm the loss of the chloramphenicol resistance cassette and of the pCP20 plasmid, each colonies were inoculated also in 5 ml of LB+Cm12.5 and 5 ml of LB+Amp.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 3rd==<br />
Glycerol stock for:<br />
*I55-1<br />
*IC-1/2/3/4-4C5<br />
<br />
----<br />
Pick of a single non-red colony of INTEIN_C3 and inoculum into 5 ml LB+Cm34. Sample was let grow ON at +37°C, 220 rpm.<br />
<br />
----<br />
Miniprep and quantification of:<br />
*I55-1: 57,4 ng/ul<br />
*I55-1bis: 79,6 ng/ul<br />
<br />
I55-1bis was digested E-S for about three hours and than gel run/cut.<br />
<br />
----<br />
<br />
Colony PCR for I74-1..6.<br />
<br />
----<br />
Gel run for PCR samples and I55 (E-S) and cut for this one.<br />
[[Image:UNIPV10_03_10_2010_I74_screen_I55_cut.jpg|thumb|200px|center|I74 screening and I55 E-S cut.]]<br />
Very bad PCR. We performed a massive PCR from colony during the night (I74-7..24), we ran it the following day.<br />
<br />
Gel extraction and quantification:<br />
*I55 (E-S): 5,0 ng/ul<br />
<br />
Ligation of I55 (E-S) with already available DNA:<br />
*I75: I55 (E-S) + I37 (E-X)<br />
*I77: I55 (E-S) + I57 (E-X)<br />
----<br />
Inoculum of I35 (last quantification after gel extraction was too poor; we will digest it again E-P) and I54 into 5 ml LB+Amp: ON growth at +37°C, 220 rpm.<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
----<br />
<br />
Each cultures of MC43_flip and MG43_flip in selective LB didn't grown, confirming the loss of pCP20 plasmid and of the chloramphenicol resistance cassette integrated into the E.coli genome.<br />
<br />
<br />
<br />
<br />
<!-- table previous next week --><br />
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<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/September/settimana4| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana1| Next week]]</td><br />
</tr><br />
</table><br />
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</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/September/settimana5Team:UNIPV-Pavia/Calendar/September/settimana52010-10-20T17:50:28Z<p>Matteo: /* October, 2nd */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana4|Week 4]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana5|Week 5]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>SEPTEMBER: WEEK 5</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==September, 27th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test27settembre|Tecan Test]]</font><br />
<br />
----<br />
<br />
MiniPrep and E-P digest for I62-a, I62-b, I62-c, I62-d, I62-e, I62-f<br />
<br />
DNA PCR for MyYeast 1-2-3<br />
<br />
Gel run<br />
[[Image:UNIPV10_27_09_2010_I62_screen.jpg|thumb|200px|center|I62 and MyYeast screening.]]<br />
<br />
<br />
I62-e was positive, so we made glycerol stock and stored it at -80°C.<br />
<br />
<br />
Protein electrophoresis for phasins.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 28th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test28settembre|Tecan Test]]</font><br />
<br />
----<br />
Miniprep and quantification for:<br />
*I1: 59,6 ng/ul<br />
*I4: 109,9 ng/ul<br />
*I53: 160,3 ng/ul<br />
*<partinfo>BBa_J04450</partinfo>-1:55,9 ng/ul<br />
*<partinfo>BBa_J04450</partinfo>-2: 47 ng/ul<br />
<br />
ON digestion of:<br />
*I1 (E-S)<br />
*I4 (E-S)<br />
*I53 (E-X)<br />
*<partinfo>BBa_J04450</partinfo>-1 (E-P)<br />
*<partinfo>BBa_J04450</partinfo>-2 (E-P)<br />
*MyYeast-3 (DNA already available) (E-S)<br />
*<partinfo>pSB4C5</partinfo> (DNA already available) (E-P)<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 29th==<br />
<br />
Colony PCR for 6 newly picked colonies.<br />
[[Image:UNIPV10_29_09_2010_I55_screen.jpg|thumb|200px|center|I55 colony PCR screening.]]<br />
No positives nor this time.<br />
----<br />
<br />
<br />
Gel run for all digested parts<br />
[[Image:UNIPV10_29_09_2010_digestions.jpg|thumb|200px|center|Digestions.]]<br />
and cut/gel purification for:<br />
*4C5 (E-P): 19,3 ng/ul<br />
*My Yeast 3 (E-S): 20,8 ng/ul<br />
*I1: 3,8 ng/ul<br />
*I4 (E-S): 14,2 ng/ul<br />
*I53 (E-X): 21,6 ng/ul<br />
*<partinfo>BBa_J04450</partinfo> (pLac-RBS-mRFP-TT) (E-P): 5,6 ng/ul<br />
*<partinfo>pSB1C3</partinfo>: 10 ng/ul<br />
<br />
<br />
Ligation of:<br />
{| align='center', border='1'<br />
| I70=MyYeast 3(E-S)+I4(E-S)<br />
|-<br />
| I71=MyYeast 3(E-S)+I1(E-S)<br />
|-<br />
|I72=<partinfo>BBa_J04450</partinfo>(E-P)+<partinfo>pSB4C5</partinfo><br />
|-<br />
|I73= I38 (E-S) + I53 (E-X)<br />
|}<br />
<br />
----<br />
<br />
Inoculum of MC43, MG43 in 5ml LB with chloramphenicol concentration of 12.5 ug/ml.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 30th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test16settembre|Tecan Test]]</font><br />
<br />
----<br />
<br />
Transformation of I73 (1ul) into ''E. coli'' TOP10. It was plated on LB+Amp 100 ug/ml agar plate.<br />
<br />
Trasformation of: <br />
*I70 (myYeast with I4, Ptef1-mOrange-tAdh1) in TOP10 and plated on LB+Amp<br />
*I71 (myYeast with I1, mOrange-tAdh1) in TOP10 and plated on LB+Amp<br />
*I72 (pLac-RFP in pSB4C5) in TOP10 and plated on LB+Cm 12,5<br />
<br />
All plates were incubated at 37°C ON<br />
<br />
<br />
Since I55 was negative again we decided to ligate it again (probably we had a bad digest for vector - I20 - and a good one for insert - we used I38 in other ligations without problems). We diegested already miniprepped DNA for three hours with 1 ul of enzymes:<br />
*I20: (E-X)<br />
<br />
Gel run/cut/extraction/quantification<br />
[[Image:UNIPV10_30_09_2010_I20_E_X.jpg|thumb|70px|center|I20 E-X digest.]]<br />
I20 E-X: 13,4 ng/ul<br />
<br />
We repeated I55 ligation using newly digested vector and already digested I38 (E-S)<br />
<br />
I55: I38 (E-S) + I20 (E-X)<br />
<br />
Inoculum into 5ml LB+Amp of:<br />
*I31<br />
*I35<br />
*I37<br />
*I57<br />
*I60<br />
*INTEIN<br />
*3x <partinfo>BBa_J04450</partinfo> (to take <partinfo>pSB1C3</partinfo>, so <partinfo>pSB1C3</partinfo> from now on)<br />
Cultures were let grow ON at 37°C, 220 rpm.<br />
<br />
----<br />
<br />
Inoculum of E. coli TOP10 in 1,5 ml LB, grown ON at 37°C 220 rpm. Tomorrow we will prepare competent cells!!<br />
<br />
Inoculum of:<br />
<br />
*S1 in 500ul M9+Cm 12,5<br />
*I47-S1 in 500ul M9+Amp+Cm 12,5<br />
*GFP-S1 in 500ul M9+Amp+Cm 12,5<br />
*RBS32 in 500ul M9+Amp<br />
<br />
Cultures were grown ON at 37°C 220 rpm.<br />
<br />
Tomorrow we will test the ability of these strains to produce GFP and to lyse when HSL is added.<br />
<br />
----<br />
In order to excise the chloramphenicol resistance cassette from the integrants in E.coli genome we transformed the cells with a plasmid (pCP20 plasmid) carrying the gene encoding for the flippase protein. This protein is able to cut fragment between two FRT flanking sequences. pCP20 was transformed in MC43-a and MG43-a and plated on LB+Amp agar plate.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 1st==<br />
<br />
This morning, we checked tranformed plates:<br />
<br />
*I72 showed NO colony!! :/ we will repeat this ligation next week<br />
*I70 and I71 showed colonies, 5 for each plate were picked and inoculated in 1ml LB+Amp to prepare glycerol stocks and for screening.<br />
*I73 showed colonies; six of them were picked and inoculated into 5 ml LB+Amp and let grow ON at 37°C, 220rpm in order to do E-P screeening the following day.<br />
<br />
----<br />
<br />
Transformation of I55 into ''E. coli'' DH5-alpha. It was plated on LB+Amp 100 agar plate and let grow ON at +37°C.<br />
<br />
----<br />
<br />
Miniprep and Nanodrop quantification for:<br />
*I31: 76,1 ng/ul<br />
*I35: 66,8 ng/ul<br />
*I60: 279,7 ng/ul<br />
(they were also sent sequencing)<br />
*INTEIN: 123,8 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-1: 104,8 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-2: 141,2 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-3: 113,5 ng/ul<br />
*I37: 174,9 ng/ul<br />
<br />
Digest:<br />
*<partinfo>pSB1C3</partinfo>: E-P<br />
*<partinfo>pSB1C3</partinfo>: E-S<br />
*<partinfo>pSB1C3</partinfo>: X-P<br />
*INTEIN: E-P<br />
*I60: E-S<br />
*I31: S-P<br />
*I35: E-P<br />
*I35: E-X<br />
*I37: E-X<br />
*I57: E-X<br />
<br />
Gel run/cut/extraction/quantification:<br />
{|align="center"<br />
|[[Image:UNIPV10_01_10_2010_digestioni_medio.jpg|thumb|200px|center|Intein, I31, I35, I37, I57, I60 digestions]] || [[Image:UNIPV10_01_10_2010_digestioni_psb1c3.jpg|thumb|150px|center|<partinfo>pSB1C3</partinfo> digested E-P (both vector and insert were kept), E-S, X-P.]]<br />
|}<br />
<br />
*I37 (E-X): 20,4 ng/ul<br />
*I57 (E-X): 17,6 ng/ul<br />
*I60 (E-S): 9,5 ng/ul<br />
They were stored at -20°C.<br />
*I31 (E-S): 5,4 ng/ul<br />
*I35 (E-P): 1,0 ng/ul ;-(<br />
*I35 (E-X): 14,4 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (E-P): 14,3 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (E-S): 15,2 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (X-P): 13,9 ng/ul<br />
<br />
ON ligation of:<br />
*I74: I31 (E-S) + I35 (E-X)<br />
*INTEIN_C3: INTEIN (E-P) + <partinfo>pSB1C3</partinfo> (E-P)<br />
<br />
----<br />
<br />
Single colony was picked from MC43 and MG43 plates and inoculated in 5 ml of LB and incubated at 37°C, 220 rpm ON.<br />
12 hours late 5 ul were streaked on non selective LB plates and incubated at 43°C ON in order to ensure the loss of pCP20 plasmid.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 2nd==<br />
<br />
PCR from colony for I55 (eleven colonies were picked).<br />
<br />
Gel run:<br />
<br />
[[Image:UNIPV10_02_10_2010_I55_screening.jpg|thumb|200px|center|I55 colony PCR screening.]]<br />
<br />
We decided to make glycerol stock for I55-1. Inoculum into 5 ml LB+Amp of the positive culture that was let grow ON at 37°C, 220 rpm.<br />
----<br />
Miniprep and quantification for I73-1/2/3/4/5/6:<br />
*I73-1: 154 ng/ul<br />
*I73-2: 177 ng/ul<br />
*I73-3: 154,7 ng/ul<br />
*I73-4: 145,5ng/ul<br />
*I73-5: 152,6 ng/ul<br />
*I73-6: 154,2 ng/ul<br />
1,5 hours E-P digest (for screening) and gel run<br />
<br />
[[Image:UNIPV10_02_10_2010_I73_screening.jpg|thumb|200px|center|I70, I71, I73, A9_4C5, A99_4C5 screening.]]<br />
<br />
As you can see they are (except for I73-6) all positive. So we stocked I73-1 and stored it at -80°C.<br />
<br />
----<br />
Transformation of I74 and INTEIN_C3 (1ul) in ''E. coli'' TOP10. They were plated on LB+Amp100 and LB+Cm34 agar plates respectively. They were let grow ON at +37°C.<br />
<br />
----<br />
Inoculum of I55-1 into 5 ml LB+Amp. ON growth at +37°C, 220 rpm.<br />
<br />
----<br />
<br />
3 colonies each plates were picked from MC43_flip and MG43_flip plates and inoculated in 5 ml of non selective LB. To confirm the loss of the chloramphenicol resistance cassette and of the pCP20 plasmid, each colonies were inoculated also in 5 ml of LB+Cm12.5 and 5 ml of LB+Amp.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 3rd==<br />
Glycerol stock for:<br />
*I55-1<br />
*IC-1/2/3/4-4C5<br />
<br />
----<br />
Pick of a single non-red colony of INTEIN_C3 and inoculum into 5 ml LB+Cm34. Sample was let grow ON at +37°C, 220 rpm.<br />
<br />
----<br />
Miniprep and quantification of:<br />
*I55-1: 57,4 ng/ul<br />
*I55-1bis: 79,6 ng/ul<br />
<br />
I55-1bis was digested E-S for about three hours and than gel run/cut.<br />
<br />
----<br />
<br />
Colony PCR for I74-1..6.<br />
<br />
----<br />
Gel run for PCR samples and I55 (E-S) and cut for this one.<br />
[[Image:UNIPV10_03_10_2010_I74_screen_I55_cut.jpg|thumb|200px|center|I74 screening and I55 E-S cut.]]<br />
Very bad PCR. We performed a massive PCR from colony during the night (I74-7..24), we ran it the following day.<br />
<br />
Gel extraction and quantification:<br />
*I55 (E-S): 5,0 ng/ul<br />
<br />
Ligation of I55 (E-S) with already available DNA:<br />
*I75: I55 (E-S) + I37 (E-X)<br />
*I77: I55 (E-S) + I57 (E-X)<br />
----<br />
Inoculum of I35 (last quantification after gel extraction was too poor; we will digest it again E-P) and I54 into 5 ml LB+Amp: ON growth at +37°C, 220 rpm.<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
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<td align="left">[[Team:UNIPV-Pavia/Calendar/September/settimana4| Previous week]]</td><br />
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<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
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</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/September/settimana5Team:UNIPV-Pavia/Calendar/September/settimana52010-10-20T17:45:47Z<p>Matteo: /* September, 30th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
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{{UNIPV-Pavia/Style}}<br />
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<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
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<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana4|Week 4]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana5|Week 5]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>SEPTEMBER: WEEK 5</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==September, 27th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test27settembre|Tecan Test]]</font><br />
<br />
----<br />
<br />
MiniPrep and E-P digest for I62-a, I62-b, I62-c, I62-d, I62-e, I62-f<br />
<br />
DNA PCR for MyYeast 1-2-3<br />
<br />
Gel run<br />
[[Image:UNIPV10_27_09_2010_I62_screen.jpg|thumb|200px|center|I62 and MyYeast screening.]]<br />
<br />
<br />
I62-e was positive, so we made glycerol stock and stored it at -80°C.<br />
<br />
<br />
Protein electrophoresis for phasins.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 28th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test28settembre|Tecan Test]]</font><br />
<br />
----<br />
Miniprep and quantification for:<br />
*I1: 59,6 ng/ul<br />
*I4: 109,9 ng/ul<br />
*I53: 160,3 ng/ul<br />
*<partinfo>BBa_J04450</partinfo>-1:55,9 ng/ul<br />
*<partinfo>BBa_J04450</partinfo>-2: 47 ng/ul<br />
<br />
ON digestion of:<br />
*I1 (E-S)<br />
*I4 (E-S)<br />
*I53 (E-X)<br />
*<partinfo>BBa_J04450</partinfo>-1 (E-P)<br />
*<partinfo>BBa_J04450</partinfo>-2 (E-P)<br />
*MyYeast-3 (DNA already available) (E-S)<br />
*<partinfo>pSB4C5</partinfo> (DNA already available) (E-P)<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 29th==<br />
<br />
Colony PCR for 6 newly picked colonies.<br />
[[Image:UNIPV10_29_09_2010_I55_screen.jpg|thumb|200px|center|I55 colony PCR screening.]]<br />
No positives nor this time.<br />
----<br />
<br />
<br />
Gel run for all digested parts<br />
[[Image:UNIPV10_29_09_2010_digestions.jpg|thumb|200px|center|Digestions.]]<br />
and cut/gel purification for:<br />
*4C5 (E-P): 19,3 ng/ul<br />
*My Yeast 3 (E-S): 20,8 ng/ul<br />
*I1: 3,8 ng/ul<br />
*I4 (E-S): 14,2 ng/ul<br />
*I53 (E-X): 21,6 ng/ul<br />
*<partinfo>BBa_J04450</partinfo> (pLac-RBS-mRFP-TT) (E-P): 5,6 ng/ul<br />
*<partinfo>pSB1C3</partinfo>: 10 ng/ul<br />
<br />
<br />
Ligation of:<br />
{| align='center', border='1'<br />
| I70=MyYeast 3(E-S)+I4(E-S)<br />
|-<br />
| I71=MyYeast 3(E-S)+I1(E-S)<br />
|-<br />
|I72=<partinfo>BBa_J04450</partinfo>(E-P)+<partinfo>pSB4C5</partinfo><br />
|-<br />
|I73= I38 (E-S) + I53 (E-X)<br />
|}<br />
<br />
----<br />
<br />
Inoculum of MC43, MG43 in 5ml LB with chloramphenicol concentration of 12.5 ug/ml.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 30th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test16settembre|Tecan Test]]</font><br />
<br />
----<br />
<br />
Transformation of I73 (1ul) into ''E. coli'' TOP10. It was plated on LB+Amp 100 ug/ml agar plate.<br />
<br />
Trasformation of: <br />
*I70 (myYeast with I4, Ptef1-mOrange-tAdh1) in TOP10 and plated on LB+Amp<br />
*I71 (myYeast with I1, mOrange-tAdh1) in TOP10 and plated on LB+Amp<br />
*I72 (pLac-RFP in pSB4C5) in TOP10 and plated on LB+Cm 12,5<br />
<br />
All plates were incubated at 37°C ON<br />
<br />
<br />
Since I55 was negative again we decided to ligate it again (probably we had a bad digest for vector - I20 - and a good one for insert - we used I38 in other ligations without problems). We diegested already miniprepped DNA for three hours with 1 ul of enzymes:<br />
*I20: (E-X)<br />
<br />
Gel run/cut/extraction/quantification<br />
[[Image:UNIPV10_30_09_2010_I20_E_X.jpg|thumb|70px|center|I20 E-X digest.]]<br />
I20 E-X: 13,4 ng/ul<br />
<br />
We repeated I55 ligation using newly digested vector and already digested I38 (E-S)<br />
<br />
I55: I38 (E-S) + I20 (E-X)<br />
<br />
Inoculum into 5ml LB+Amp of:<br />
*I31<br />
*I35<br />
*I37<br />
*I57<br />
*I60<br />
*INTEIN<br />
*3x <partinfo>BBa_J04450</partinfo> (to take <partinfo>pSB1C3</partinfo>, so <partinfo>pSB1C3</partinfo> from now on)<br />
Cultures were let grow ON at 37°C, 220 rpm.<br />
<br />
----<br />
<br />
Inoculum of E. coli TOP10 in 1,5 ml LB, grown ON at 37°C 220 rpm. Tomorrow we will prepare competent cells!!<br />
<br />
Inoculum of:<br />
<br />
*S1 in 500ul M9+Cm 12,5<br />
*I47-S1 in 500ul M9+Amp+Cm 12,5<br />
*GFP-S1 in 500ul M9+Amp+Cm 12,5<br />
*RBS32 in 500ul M9+Amp<br />
<br />
Cultures were grown ON at 37°C 220 rpm.<br />
<br />
Tomorrow we will test the ability of these strains to produce GFP and to lyse when HSL is added.<br />
<br />
----<br />
In order to excise the chloramphenicol resistance cassette from the integrants in E.coli genome we transformed the cells with a plasmid (pCP20 plasmid) carrying the gene encoding for the flippase protein. This protein is able to cut fragment between two FRT flanking sequences. pCP20 was transformed in MC43-a and MG43-a and plated on LB+Amp agar plate.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 1st==<br />
<br />
This morning, we checked tranformed plates:<br />
<br />
*I72 showed NO colony!! :/ we will repeat this ligation next week<br />
*I70 and I71 showed colonies, 5 for each plate were picked and inoculated in 1ml LB+Amp to prepare glycerol stocks and for screening.<br />
*I73 showed colonies; six of them were picked and inoculated into 5 ml LB+Amp and let grow ON at 37°C, 220rpm in order to do E-P screeening the following day.<br />
<br />
----<br />
<br />
Transformation of I55 into ''E. coli'' DH5-alpha. It was plated on LB+Amp 100 agar plate and let grow ON at +37°C.<br />
<br />
----<br />
<br />
Miniprep and Nanodrop quantification for:<br />
*I31: 76,1 ng/ul<br />
*I35: 66,8 ng/ul<br />
*I60: 279,7 ng/ul<br />
(they were also sent sequencing)<br />
*INTEIN: 123,8 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-1: 104,8 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-2: 141,2 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-3: 113,5 ng/ul<br />
*I37: 174,9 ng/ul<br />
<br />
Digest:<br />
*<partinfo>pSB1C3</partinfo>: E-P<br />
*<partinfo>pSB1C3</partinfo>: E-S<br />
*<partinfo>pSB1C3</partinfo>: X-P<br />
*INTEIN: E-P<br />
*I60: E-S<br />
*I31: S-P<br />
*I35: E-P<br />
*I35: E-X<br />
*I37: E-X<br />
*I57: E-X<br />
<br />
Gel run/cut/extraction/quantification:<br />
{|align="center"<br />
|[[Image:UNIPV10_01_10_2010_digestioni_medio.jpg|thumb|200px|center|Intein, I31, I35, I37, I57, I60 digestions]] || [[Image:UNIPV10_01_10_2010_digestioni_psb1c3.jpg|thumb|150px|center|<partinfo>pSB1C3</partinfo> digested E-P (both vector and insert were kept), E-S, X-P.]]<br />
|}<br />
<br />
*I37 (E-X): 20,4 ng/ul<br />
*I57 (E-X): 17,6 ng/ul<br />
*I60 (E-S): 9,5 ng/ul<br />
They were stored at -20°C.<br />
*I31 (E-S): 5,4 ng/ul<br />
*I35 (E-P): 1,0 ng/ul ;-(<br />
*I35 (E-X): 14,4 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (E-P): 14,3 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (E-S): 15,2 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (X-P): 13,9 ng/ul<br />
<br />
ON ligation of:<br />
*I74: I31 (E-S) + I35 (E-X)<br />
*INTEIN_C3: INTEIN (E-P) + <partinfo>pSB1C3</partinfo> (E-P)<br />
<br />
----<br />
<br />
Single colony was picked from MC43 and MG43 plates and inoculated in 5 ml of LB and incubated at 37°C, 220 rpm ON.<br />
12 hours late 5 ul were streaked on non selective LB plates and incubated at 43°C ON in order to ensure the loss of pCP20 plasmid.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 2nd==<br />
<br />
PCR from colony for I55 (eleven colonies were picked).<br />
<br />
Gel run:<br />
<br />
[[Image:UNIPV10_02_10_2010_I55_screening.jpg|thumb|200px|center|I55 colony PCR screening.]]<br />
<br />
We decided to make glycerol stock for I55-1. Inoculum into 5 ml LB+Amp of the positive culture that was let grow ON at 37°C, 220 rpm.<br />
----<br />
Miniprep and quantification for I73-1/2/3/4/5/6:<br />
*I73-1: 154 ng/ul<br />
*I73-2: 177 ng/ul<br />
*I73-3: 154,7 ng/ul<br />
*I73-4: 145,5ng/ul<br />
*I73-5: 152,6 ng/ul<br />
*I73-6: 154,2 ng/ul<br />
1,5 hours E-P digest (for screening) and gel run<br />
<br />
[[Image:UNIPV10_02_10_2010_I73_screening.jpg|thumb|200px|center|I70, I71, I73, A9_4C5, A99_4C5 screening.]]<br />
<br />
As you can see they are (except for I73-6) all positive. So we stocked I73-1 and stored it at -80°C.<br />
<br />
----<br />
Transformation of I74 and INTEIN_C3 (1ul) in ''E. coli'' TOP10. They were plated on LB+Amp100 and LB+Cm34 agar plates respectively. They were let grow ON at +37°C.<br />
<br />
----<br />
Inoculum of I55-1 into 5 ml LB+Amp. ON growth at +37°C, 220 rpm.<br />
<br />
----<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 3rd==<br />
Glycerol stock for:<br />
*I55-1<br />
*IC-1/2/3/4-4C5<br />
<br />
----<br />
Pick of a single non-red colony of INTEIN_C3 and inoculum into 5 ml LB+Cm34. Sample was let grow ON at +37°C, 220 rpm.<br />
<br />
----<br />
Miniprep and quantification of:<br />
*I55-1: 57,4 ng/ul<br />
*I55-1bis: 79,6 ng/ul<br />
<br />
I55-1bis was digested E-S for about three hours and than gel run/cut.<br />
<br />
----<br />
<br />
Colony PCR for I74-1..6.<br />
<br />
----<br />
Gel run for PCR samples and I55 (E-S) and cut for this one.<br />
[[Image:UNIPV10_03_10_2010_I74_screen_I55_cut.jpg|thumb|200px|center|I74 screening and I55 E-S cut.]]<br />
Very bad PCR. We performed a massive PCR from colony during the night (I74-7..24), we ran it the following day.<br />
<br />
Gel extraction and quantification:<br />
*I55 (E-S): 5,0 ng/ul<br />
<br />
Ligation of I55 (E-S) with already available DNA:<br />
*I75: I55 (E-S) + I37 (E-X)<br />
*I77: I55 (E-S) + I57 (E-X)<br />
----<br />
Inoculum of I35 (last quantification after gel extraction was too poor; we will digest it again E-P) and I54 into 5 ml LB+Amp: ON growth at +37°C, 220 rpm.<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
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<td align="left">[[Team:UNIPV-Pavia/Calendar/September/settimana4| Previous week]]</td><br />
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{{UNIPV-Pavia/menu_mesi}}<br />
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</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/September/settimana5Team:UNIPV-Pavia/Calendar/September/settimana52010-10-20T17:43:57Z<p>Matteo: /* October, 2nd */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana4|Week 4]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana5|Week 5]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>SEPTEMBER: WEEK 5</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==September, 27th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test27settembre|Tecan Test]]</font><br />
<br />
----<br />
<br />
MiniPrep and E-P digest for I62-a, I62-b, I62-c, I62-d, I62-e, I62-f<br />
<br />
DNA PCR for MyYeast 1-2-3<br />
<br />
Gel run<br />
[[Image:UNIPV10_27_09_2010_I62_screen.jpg|thumb|200px|center|I62 and MyYeast screening.]]<br />
<br />
<br />
I62-e was positive, so we made glycerol stock and stored it at -80°C.<br />
<br />
<br />
Protein electrophoresis for phasins.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 28th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test28settembre|Tecan Test]]</font><br />
<br />
----<br />
Miniprep and quantification for:<br />
*I1: 59,6 ng/ul<br />
*I4: 109,9 ng/ul<br />
*I53: 160,3 ng/ul<br />
*<partinfo>BBa_J04450</partinfo>-1:55,9 ng/ul<br />
*<partinfo>BBa_J04450</partinfo>-2: 47 ng/ul<br />
<br />
ON digestion of:<br />
*I1 (E-S)<br />
*I4 (E-S)<br />
*I53 (E-X)<br />
*<partinfo>BBa_J04450</partinfo>-1 (E-P)<br />
*<partinfo>BBa_J04450</partinfo>-2 (E-P)<br />
*MyYeast-3 (DNA already available) (E-S)<br />
*<partinfo>pSB4C5</partinfo> (DNA already available) (E-P)<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 29th==<br />
<br />
Colony PCR for 6 newly picked colonies.<br />
[[Image:UNIPV10_29_09_2010_I55_screen.jpg|thumb|200px|center|I55 colony PCR screening.]]<br />
No positives nor this time.<br />
----<br />
<br />
<br />
Gel run for all digested parts<br />
[[Image:UNIPV10_29_09_2010_digestions.jpg|thumb|200px|center|Digestions.]]<br />
and cut/gel purification for:<br />
*4C5 (E-P): 19,3 ng/ul<br />
*My Yeast 3 (E-S): 20,8 ng/ul<br />
*I1: 3,8 ng/ul<br />
*I4 (E-S): 14,2 ng/ul<br />
*I53 (E-X): 21,6 ng/ul<br />
*<partinfo>BBa_J04450</partinfo> (pLac-RBS-mRFP-TT) (E-P): 5,6 ng/ul<br />
*<partinfo>pSB1C3</partinfo>: 10 ng/ul<br />
<br />
<br />
Ligation of:<br />
{| align='center', border='1'<br />
| I70=MyYeast 3(E-S)+I4(E-S)<br />
|-<br />
| I71=MyYeast 3(E-S)+I1(E-S)<br />
|-<br />
|I72=<partinfo>BBa_J04450</partinfo>(E-P)+<partinfo>pSB4C5</partinfo><br />
|-<br />
|I73= I38 (E-S) + I53 (E-X)<br />
|}<br />
<br />
----<br />
<br />
Inoculum of MC43, MG43 in 5ml LB with chloramphenicol concentration of 12.5 ug/ml.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 30th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test16settembre|Tecan Test]]</font><br />
<br />
----<br />
<br />
Transformation of I73 (1ul) into ''E. coli'' TOP10. It was plated on LB+Amp 100 ug/ml agar plate.<br />
<br />
Trasformation of: <br />
*I70 (myYeast with I4, Ptef1-mOrange-tAdh1) in TOP10 and plated on LB+Amp<br />
*I71 (myYeast with I1, mOrange-tAdh1) in TOP10 and plated on LB+Amp<br />
*I72 (pLac-RFP in pSB4C5) in TOP10 and plated on LB+Cm 12,5<br />
<br />
All plates were incubated at 37°C ON<br />
<br />
<br />
Since I55 was negative again we decided to ligate it again (probably we had a bad digest for vector - I20 - and a good one for insert - we used I38 in other ligations without problems). We diegested already miniprepped DNA for three hours with 1 ul of enzymes:<br />
*I20: (E-X)<br />
<br />
Gel run/cut/extraction/quantification<br />
[[Image:UNIPV10_30_09_2010_I20_E_X.jpg|thumb|70px|center|I20 E-X digest.]]<br />
I20 E-X: 13,4 ng/ul<br />
<br />
We repeated I55 ligation using newly digested vector and already digested I38 (E-S)<br />
<br />
I55: I38 (E-S) + I20 (E-X)<br />
<br />
Inoculum into 5ml LB+Amp of:<br />
*I31<br />
*I35<br />
*I37<br />
*I57<br />
*I60<br />
*INTEIN<br />
*3x <partinfo>BBa_J04450</partinfo> (to take <partinfo>pSB1C3</partinfo>, so <partinfo>pSB1C3</partinfo> from now on)<br />
Cultures were let grow ON at 37°C, 220 rpm.<br />
<br />
----<br />
<br />
Inoculum of E. coli TOP10 in 1,5 ml LB, grown ON at 37°C 220 rpm. Tomorrow we will prepare competent cells!!<br />
<br />
Inoculum of:<br />
<br />
*S1 in 500ul M9+Cm 12,5<br />
*I47-S1 in 500ul M9+Amp+Cm 12,5<br />
*GFP-S1 in 500ul M9+Amp+Cm 12,5<br />
*RBS32 in 500ul M9+Amp<br />
<br />
Cultures were grown ON at 37°C 220 rpm.<br />
<br />
Tomorrow we will test the ability of these strains to produce GFP and to lyse when HSL is added.<br />
<br />
----<br />
In order to excise the chloramphenicol resistance cassette from the integrants in E.coli genome we transformed the cells with a plasmid (pCP20 plasmid) carrying the gene encoding for the flippase protein. This protein is able to cut fragment between two FRT flanking sequences. pCP20 was transformed in MC43-abc and MG43-abc and plated on LB+Amp agar plate.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 1st==<br />
<br />
This morning, we checked tranformed plates:<br />
<br />
*I72 showed NO colony!! :/ we will repeat this ligation next week<br />
*I70 and I71 showed colonies, 5 for each plate were picked and inoculated in 1ml LB+Amp to prepare glycerol stocks and for screening.<br />
*I73 showed colonies; six of them were picked and inoculated into 5 ml LB+Amp and let grow ON at 37°C, 220rpm in order to do E-P screeening the following day.<br />
<br />
----<br />
<br />
Transformation of I55 into ''E. coli'' DH5-alpha. It was plated on LB+Amp 100 agar plate and let grow ON at +37°C.<br />
<br />
----<br />
<br />
Miniprep and Nanodrop quantification for:<br />
*I31: 76,1 ng/ul<br />
*I35: 66,8 ng/ul<br />
*I60: 279,7 ng/ul<br />
(they were also sent sequencing)<br />
*INTEIN: 123,8 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-1: 104,8 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-2: 141,2 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-3: 113,5 ng/ul<br />
*I37: 174,9 ng/ul<br />
<br />
Digest:<br />
*<partinfo>pSB1C3</partinfo>: E-P<br />
*<partinfo>pSB1C3</partinfo>: E-S<br />
*<partinfo>pSB1C3</partinfo>: X-P<br />
*INTEIN: E-P<br />
*I60: E-S<br />
*I31: S-P<br />
*I35: E-P<br />
*I35: E-X<br />
*I37: E-X<br />
*I57: E-X<br />
<br />
Gel run/cut/extraction/quantification:<br />
{|align="center"<br />
|[[Image:UNIPV10_01_10_2010_digestioni_medio.jpg|thumb|200px|center|Intein, I31, I35, I37, I57, I60 digestions]] || [[Image:UNIPV10_01_10_2010_digestioni_psb1c3.jpg|thumb|150px|center|<partinfo>pSB1C3</partinfo> digested E-P (both vector and insert were kept), E-S, X-P.]]<br />
|}<br />
<br />
*I37 (E-X): 20,4 ng/ul<br />
*I57 (E-X): 17,6 ng/ul<br />
*I60 (E-S): 9,5 ng/ul<br />
They were stored at -20°C.<br />
*I31 (E-S): 5,4 ng/ul<br />
*I35 (E-P): 1,0 ng/ul ;-(<br />
*I35 (E-X): 14,4 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (E-P): 14,3 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (E-S): 15,2 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (X-P): 13,9 ng/ul<br />
<br />
ON ligation of:<br />
*I74: I31 (E-S) + I35 (E-X)<br />
*INTEIN_C3: INTEIN (E-P) + <partinfo>pSB1C3</partinfo> (E-P)<br />
<br />
----<br />
<br />
Single colony was picked from MC43 and MG43 plates and inoculated in 5 ml of LB and incubated at 37°C, 220 rpm ON.<br />
12 hours late 5 ul were streaked on non selective LB plates and incubated at 43°C ON in order to ensure the loss of pCP20 plasmid.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 2nd==<br />
<br />
PCR from colony for I55 (eleven colonies were picked).<br />
<br />
Gel run:<br />
<br />
[[Image:UNIPV10_02_10_2010_I55_screening.jpg|thumb|200px|center|I55 colony PCR screening.]]<br />
<br />
We decided to make glycerol stock for I55-1. Inoculum into 5 ml LB+Amp of the positive culture that was let grow ON at 37°C, 220 rpm.<br />
----<br />
Miniprep and quantification for I73-1/2/3/4/5/6:<br />
*I73-1: 154 ng/ul<br />
*I73-2: 177 ng/ul<br />
*I73-3: 154,7 ng/ul<br />
*I73-4: 145,5ng/ul<br />
*I73-5: 152,6 ng/ul<br />
*I73-6: 154,2 ng/ul<br />
1,5 hours E-P digest (for screening) and gel run<br />
<br />
[[Image:UNIPV10_02_10_2010_I73_screening.jpg|thumb|200px|center|I70, I71, I73, A9_4C5, A99_4C5 screening.]]<br />
<br />
As you can see they are (except for I73-6) all positive. So we stocked I73-1 and stored it at -80°C.<br />
<br />
----<br />
Transformation of I74 and INTEIN_C3 (1ul) in ''E. coli'' TOP10. They were plated on LB+Amp100 and LB+Cm34 agar plates respectively. They were let grow ON at +37°C.<br />
<br />
----<br />
Inoculum of I55-1 into 5 ml LB+Amp. ON growth at +37°C, 220 rpm.<br />
<br />
----<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 3rd==<br />
Glycerol stock for:<br />
*I55-1<br />
*IC-1/2/3/4-4C5<br />
<br />
----<br />
Pick of a single non-red colony of INTEIN_C3 and inoculum into 5 ml LB+Cm34. Sample was let grow ON at +37°C, 220 rpm.<br />
<br />
----<br />
Miniprep and quantification of:<br />
*I55-1: 57,4 ng/ul<br />
*I55-1bis: 79,6 ng/ul<br />
<br />
I55-1bis was digested E-S for about three hours and than gel run/cut.<br />
<br />
----<br />
<br />
Colony PCR for I74-1..6.<br />
<br />
----<br />
Gel run for PCR samples and I55 (E-S) and cut for this one.<br />
[[Image:UNIPV10_03_10_2010_I74_screen_I55_cut.jpg|thumb|200px|center|I74 screening and I55 E-S cut.]]<br />
Very bad PCR. We performed a massive PCR from colony during the night (I74-7..24), we ran it the following day.<br />
<br />
Gel extraction and quantification:<br />
*I55 (E-S): 5,0 ng/ul<br />
<br />
Ligation of I55 (E-S) with already available DNA:<br />
*I75: I55 (E-S) + I37 (E-X)<br />
*I77: I55 (E-S) + I57 (E-X)<br />
----<br />
Inoculum of I35 (last quantification after gel extraction was too poor; we will digest it again E-P) and I54 into 5 ml LB+Amp: ON growth at +37°C, 220 rpm.<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
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<table border="0" width="100%" class="menu"><br />
<tr><br />
<td align="left">[[Team:UNIPV-Pavia/Calendar/September/settimana4| Previous week]]</td><br />
<td align="right">[[Team:UNIPV-Pavia/Calendar/October/settimana1| Next week]]</td><br />
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</td><br />
<br />
<td width="15%" align="right" valign="top"><br />
<br />
{{UNIPV-Pavia/menu_mesi}}<br />
<br />
</td><br />
</tr><br />
</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/September/settimana5Team:UNIPV-Pavia/Calendar/September/settimana52010-10-20T17:40:54Z<p>Matteo: /* October, 1st */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
</tr><br />
<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana4|Week 4]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana5|Week 5]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>SEPTEMBER: WEEK 5</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==September, 27th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test27settembre|Tecan Test]]</font><br />
<br />
----<br />
<br />
MiniPrep and E-P digest for I62-a, I62-b, I62-c, I62-d, I62-e, I62-f<br />
<br />
DNA PCR for MyYeast 1-2-3<br />
<br />
Gel run<br />
[[Image:UNIPV10_27_09_2010_I62_screen.jpg|thumb|200px|center|I62 and MyYeast screening.]]<br />
<br />
<br />
I62-e was positive, so we made glycerol stock and stored it at -80°C.<br />
<br />
<br />
Protein electrophoresis for phasins.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 28th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test28settembre|Tecan Test]]</font><br />
<br />
----<br />
Miniprep and quantification for:<br />
*I1: 59,6 ng/ul<br />
*I4: 109,9 ng/ul<br />
*I53: 160,3 ng/ul<br />
*<partinfo>BBa_J04450</partinfo>-1:55,9 ng/ul<br />
*<partinfo>BBa_J04450</partinfo>-2: 47 ng/ul<br />
<br />
ON digestion of:<br />
*I1 (E-S)<br />
*I4 (E-S)<br />
*I53 (E-X)<br />
*<partinfo>BBa_J04450</partinfo>-1 (E-P)<br />
*<partinfo>BBa_J04450</partinfo>-2 (E-P)<br />
*MyYeast-3 (DNA already available) (E-S)<br />
*<partinfo>pSB4C5</partinfo> (DNA already available) (E-P)<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 29th==<br />
<br />
Colony PCR for 6 newly picked colonies.<br />
[[Image:UNIPV10_29_09_2010_I55_screen.jpg|thumb|200px|center|I55 colony PCR screening.]]<br />
No positives nor this time.<br />
----<br />
<br />
<br />
Gel run for all digested parts<br />
[[Image:UNIPV10_29_09_2010_digestions.jpg|thumb|200px|center|Digestions.]]<br />
and cut/gel purification for:<br />
*4C5 (E-P): 19,3 ng/ul<br />
*My Yeast 3 (E-S): 20,8 ng/ul<br />
*I1: 3,8 ng/ul<br />
*I4 (E-S): 14,2 ng/ul<br />
*I53 (E-X): 21,6 ng/ul<br />
*<partinfo>BBa_J04450</partinfo> (pLac-RBS-mRFP-TT) (E-P): 5,6 ng/ul<br />
*<partinfo>pSB1C3</partinfo>: 10 ng/ul<br />
<br />
<br />
Ligation of:<br />
{| align='center', border='1'<br />
| I70=MyYeast 3(E-S)+I4(E-S)<br />
|-<br />
| I71=MyYeast 3(E-S)+I1(E-S)<br />
|-<br />
|I72=<partinfo>BBa_J04450</partinfo>(E-P)+<partinfo>pSB4C5</partinfo><br />
|-<br />
|I73= I38 (E-S) + I53 (E-X)<br />
|}<br />
<br />
----<br />
<br />
Inoculum of MC43, MG43 in 5ml LB with chloramphenicol concentration of 12.5 ug/ml.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 30th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test16settembre|Tecan Test]]</font><br />
<br />
----<br />
<br />
Transformation of I73 (1ul) into ''E. coli'' TOP10. It was plated on LB+Amp 100 ug/ml agar plate.<br />
<br />
Trasformation of: <br />
*I70 (myYeast with I4, Ptef1-mOrange-tAdh1) in TOP10 and plated on LB+Amp<br />
*I71 (myYeast with I1, mOrange-tAdh1) in TOP10 and plated on LB+Amp<br />
*I72 (pLac-RFP in pSB4C5) in TOP10 and plated on LB+Cm 12,5<br />
<br />
All plates were incubated at 37°C ON<br />
<br />
<br />
Since I55 was negative again we decided to ligate it again (probably we had a bad digest for vector - I20 - and a good one for insert - we used I38 in other ligations without problems). We diegested already miniprepped DNA for three hours with 1 ul of enzymes:<br />
*I20: (E-X)<br />
<br />
Gel run/cut/extraction/quantification<br />
[[Image:UNIPV10_30_09_2010_I20_E_X.jpg|thumb|70px|center|I20 E-X digest.]]<br />
I20 E-X: 13,4 ng/ul<br />
<br />
We repeated I55 ligation using newly digested vector and already digested I38 (E-S)<br />
<br />
I55: I38 (E-S) + I20 (E-X)<br />
<br />
Inoculum into 5ml LB+Amp of:<br />
*I31<br />
*I35<br />
*I37<br />
*I57<br />
*I60<br />
*INTEIN<br />
*3x <partinfo>BBa_J04450</partinfo> (to take <partinfo>pSB1C3</partinfo>, so <partinfo>pSB1C3</partinfo> from now on)<br />
Cultures were let grow ON at 37°C, 220 rpm.<br />
<br />
----<br />
<br />
Inoculum of E. coli TOP10 in 1,5 ml LB, grown ON at 37°C 220 rpm. Tomorrow we will prepare competent cells!!<br />
<br />
Inoculum of:<br />
<br />
*S1 in 500ul M9+Cm 12,5<br />
*I47-S1 in 500ul M9+Amp+Cm 12,5<br />
*GFP-S1 in 500ul M9+Amp+Cm 12,5<br />
*RBS32 in 500ul M9+Amp<br />
<br />
Cultures were grown ON at 37°C 220 rpm.<br />
<br />
Tomorrow we will test the ability of these strains to produce GFP and to lyse when HSL is added.<br />
<br />
----<br />
In order to excise the chloramphenicol resistance cassette from the integrants in E.coli genome we transformed the cells with a plasmid (pCP20 plasmid) carrying the gene encoding for the flippase protein. This protein is able to cut fragment between two FRT flanking sequences. pCP20 was transformed in MC43-abc and MG43-abc and plated on LB+Amp agar plate.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 1st==<br />
<br />
This morning, we checked tranformed plates:<br />
<br />
*I72 showed NO colony!! :/ we will repeat this ligation next week<br />
*I70 and I71 showed colonies, 5 for each plate were picked and inoculated in 1ml LB+Amp to prepare glycerol stocks and for screening.<br />
*I73 showed colonies; six of them were picked and inoculated into 5 ml LB+Amp and let grow ON at 37°C, 220rpm in order to do E-P screeening the following day.<br />
<br />
----<br />
<br />
Transformation of I55 into ''E. coli'' DH5-alpha. It was plated on LB+Amp 100 agar plate and let grow ON at +37°C.<br />
<br />
----<br />
<br />
Miniprep and Nanodrop quantification for:<br />
*I31: 76,1 ng/ul<br />
*I35: 66,8 ng/ul<br />
*I60: 279,7 ng/ul<br />
(they were also sent sequencing)<br />
*INTEIN: 123,8 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-1: 104,8 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-2: 141,2 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-3: 113,5 ng/ul<br />
*I37: 174,9 ng/ul<br />
<br />
Digest:<br />
*<partinfo>pSB1C3</partinfo>: E-P<br />
*<partinfo>pSB1C3</partinfo>: E-S<br />
*<partinfo>pSB1C3</partinfo>: X-P<br />
*INTEIN: E-P<br />
*I60: E-S<br />
*I31: S-P<br />
*I35: E-P<br />
*I35: E-X<br />
*I37: E-X<br />
*I57: E-X<br />
<br />
Gel run/cut/extraction/quantification:<br />
{|align="center"<br />
|[[Image:UNIPV10_01_10_2010_digestioni_medio.jpg|thumb|200px|center|Intein, I31, I35, I37, I57, I60 digestions]] || [[Image:UNIPV10_01_10_2010_digestioni_psb1c3.jpg|thumb|150px|center|<partinfo>pSB1C3</partinfo> digested E-P (both vector and insert were kept), E-S, X-P.]]<br />
|}<br />
<br />
*I37 (E-X): 20,4 ng/ul<br />
*I57 (E-X): 17,6 ng/ul<br />
*I60 (E-S): 9,5 ng/ul<br />
They were stored at -20°C.<br />
*I31 (E-S): 5,4 ng/ul<br />
*I35 (E-P): 1,0 ng/ul ;-(<br />
*I35 (E-X): 14,4 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (E-P): 14,3 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (E-S): 15,2 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (X-P): 13,9 ng/ul<br />
<br />
ON ligation of:<br />
*I74: I31 (E-S) + I35 (E-X)<br />
*INTEIN_C3: INTEIN (E-P) + <partinfo>pSB1C3</partinfo> (E-P)<br />
<br />
----<br />
<br />
Single colony was picked from MC43 and MG43 plates and inoculated in 5 ml of LB and incubated at 37°C, 220 rpm ON.<br />
12 hours late 5 ul were streaked on non selective LB plates and incubated at 43°C ON in order to ensure the loss of pCP20 plasmid.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 2nd==<br />
<br />
PCR from colony for I55 (eleven colonies were picked).<br />
<br />
Gel run:<br />
<br />
[[Image:UNIPV10_02_10_2010_I55_screening.jpg|thumb|200px|center|I55 colony PCR screening.]]<br />
<br />
We decided to make glycerol stock for I55-1. Inoculum into 5 ml LB+Amp of the positive culture that was let grow ON at 37°C, 220 rpm.<br />
----<br />
Miniprep and quantification for I73-1/2/3/4/5/6:<br />
*I73-1: 154 ng/ul<br />
*I73-2: 177 ng/ul<br />
*I73-3: 154,7 ng/ul<br />
*I73-4: 145,5ng/ul<br />
*I73-5: 152,6 ng/ul<br />
*I73-6: 154,2 ng/ul<br />
1,5 hours E-P digest (for screening) and gel run<br />
<br />
[[Image:UNIPV10_02_10_2010_I73_screening.jpg|thumb|200px|center|I70, I71, I73, A9_4C5, A99_4C5 screening.]]<br />
<br />
As you can see they are (except for I73-6) all positive. So we stocked I73-1 and stored it at -80°C.<br />
<br />
----<br />
Transformation of I74 and INTEIN_C3 (1ul) in ''E. coli'' TOP10. They were plated on LB+Amp100 and LB+Cm34 agar plates respectively. They were let grow ON at +37°C.<br />
<br />
----<br />
Inoculum of I55-1 into 5 ml LB+Amp. ON growth at +37°C, 220 rpm.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 3rd==<br />
Glycerol stock for:<br />
*I55-1<br />
*IC-1/2/3/4-4C5<br />
<br />
----<br />
Pick of a single non-red colony of INTEIN_C3 and inoculum into 5 ml LB+Cm34. Sample was let grow ON at +37°C, 220 rpm.<br />
<br />
----<br />
Miniprep and quantification of:<br />
*I55-1: 57,4 ng/ul<br />
*I55-1bis: 79,6 ng/ul<br />
<br />
I55-1bis was digested E-S for about three hours and than gel run/cut.<br />
<br />
----<br />
<br />
Colony PCR for I74-1..6.<br />
<br />
----<br />
Gel run for PCR samples and I55 (E-S) and cut for this one.<br />
[[Image:UNIPV10_03_10_2010_I74_screen_I55_cut.jpg|thumb|200px|center|I74 screening and I55 E-S cut.]]<br />
Very bad PCR. We performed a massive PCR from colony during the night (I74-7..24), we ran it the following day.<br />
<br />
Gel extraction and quantification:<br />
*I55 (E-S): 5,0 ng/ul<br />
<br />
Ligation of I55 (E-S) with already available DNA:<br />
*I75: I55 (E-S) + I37 (E-X)<br />
*I77: I55 (E-S) + I57 (E-X)<br />
----<br />
Inoculum of I35 (last quantification after gel extraction was too poor; we will digest it again E-P) and I54 into 5 ml LB+Amp: ON growth at +37°C, 220 rpm.<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
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</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/September/settimana5Team:UNIPV-Pavia/Calendar/September/settimana52010-10-20T17:35:42Z<p>Matteo: /* September, 30th */</p>
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<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
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<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana4|Week 4]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana5|Week 5]]<br />
</td><br />
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<br><br />
<br />
<html><p align="center"><font size="4"><b>SEPTEMBER: WEEK 5</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==September, 27th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test27settembre|Tecan Test]]</font><br />
<br />
----<br />
<br />
MiniPrep and E-P digest for I62-a, I62-b, I62-c, I62-d, I62-e, I62-f<br />
<br />
DNA PCR for MyYeast 1-2-3<br />
<br />
Gel run<br />
[[Image:UNIPV10_27_09_2010_I62_screen.jpg|thumb|200px|center|I62 and MyYeast screening.]]<br />
<br />
<br />
I62-e was positive, so we made glycerol stock and stored it at -80°C.<br />
<br />
<br />
Protein electrophoresis for phasins.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 28th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test28settembre|Tecan Test]]</font><br />
<br />
----<br />
Miniprep and quantification for:<br />
*I1: 59,6 ng/ul<br />
*I4: 109,9 ng/ul<br />
*I53: 160,3 ng/ul<br />
*<partinfo>BBa_J04450</partinfo>-1:55,9 ng/ul<br />
*<partinfo>BBa_J04450</partinfo>-2: 47 ng/ul<br />
<br />
ON digestion of:<br />
*I1 (E-S)<br />
*I4 (E-S)<br />
*I53 (E-X)<br />
*<partinfo>BBa_J04450</partinfo>-1 (E-P)<br />
*<partinfo>BBa_J04450</partinfo>-2 (E-P)<br />
*MyYeast-3 (DNA already available) (E-S)<br />
*<partinfo>pSB4C5</partinfo> (DNA already available) (E-P)<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 29th==<br />
<br />
Colony PCR for 6 newly picked colonies.<br />
[[Image:UNIPV10_29_09_2010_I55_screen.jpg|thumb|200px|center|I55 colony PCR screening.]]<br />
No positives nor this time.<br />
----<br />
<br />
<br />
Gel run for all digested parts<br />
[[Image:UNIPV10_29_09_2010_digestions.jpg|thumb|200px|center|Digestions.]]<br />
and cut/gel purification for:<br />
*4C5 (E-P): 19,3 ng/ul<br />
*My Yeast 3 (E-S): 20,8 ng/ul<br />
*I1: 3,8 ng/ul<br />
*I4 (E-S): 14,2 ng/ul<br />
*I53 (E-X): 21,6 ng/ul<br />
*<partinfo>BBa_J04450</partinfo> (pLac-RBS-mRFP-TT) (E-P): 5,6 ng/ul<br />
*<partinfo>pSB1C3</partinfo>: 10 ng/ul<br />
<br />
<br />
Ligation of:<br />
{| align='center', border='1'<br />
| I70=MyYeast 3(E-S)+I4(E-S)<br />
|-<br />
| I71=MyYeast 3(E-S)+I1(E-S)<br />
|-<br />
|I72=<partinfo>BBa_J04450</partinfo>(E-P)+<partinfo>pSB4C5</partinfo><br />
|-<br />
|I73= I38 (E-S) + I53 (E-X)<br />
|}<br />
<br />
----<br />
<br />
Inoculum of MC43, MG43 in 5ml LB with chloramphenicol concentration of 12.5 ug/ml.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 30th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test16settembre|Tecan Test]]</font><br />
<br />
----<br />
<br />
Transformation of I73 (1ul) into ''E. coli'' TOP10. It was plated on LB+Amp 100 ug/ml agar plate.<br />
<br />
Trasformation of: <br />
*I70 (myYeast with I4, Ptef1-mOrange-tAdh1) in TOP10 and plated on LB+Amp<br />
*I71 (myYeast with I1, mOrange-tAdh1) in TOP10 and plated on LB+Amp<br />
*I72 (pLac-RFP in pSB4C5) in TOP10 and plated on LB+Cm 12,5<br />
<br />
All plates were incubated at 37°C ON<br />
<br />
<br />
Since I55 was negative again we decided to ligate it again (probably we had a bad digest for vector - I20 - and a good one for insert - we used I38 in other ligations without problems). We diegested already miniprepped DNA for three hours with 1 ul of enzymes:<br />
*I20: (E-X)<br />
<br />
Gel run/cut/extraction/quantification<br />
[[Image:UNIPV10_30_09_2010_I20_E_X.jpg|thumb|70px|center|I20 E-X digest.]]<br />
I20 E-X: 13,4 ng/ul<br />
<br />
We repeated I55 ligation using newly digested vector and already digested I38 (E-S)<br />
<br />
I55: I38 (E-S) + I20 (E-X)<br />
<br />
Inoculum into 5ml LB+Amp of:<br />
*I31<br />
*I35<br />
*I37<br />
*I57<br />
*I60<br />
*INTEIN<br />
*3x <partinfo>BBa_J04450</partinfo> (to take <partinfo>pSB1C3</partinfo>, so <partinfo>pSB1C3</partinfo> from now on)<br />
Cultures were let grow ON at 37°C, 220 rpm.<br />
<br />
----<br />
<br />
Inoculum of E. coli TOP10 in 1,5 ml LB, grown ON at 37°C 220 rpm. Tomorrow we will prepare competent cells!!<br />
<br />
Inoculum of:<br />
<br />
*S1 in 500ul M9+Cm 12,5<br />
*I47-S1 in 500ul M9+Amp+Cm 12,5<br />
*GFP-S1 in 500ul M9+Amp+Cm 12,5<br />
*RBS32 in 500ul M9+Amp<br />
<br />
Cultures were grown ON at 37°C 220 rpm.<br />
<br />
Tomorrow we will test the ability of these strains to produce GFP and to lyse when HSL is added.<br />
<br />
----<br />
In order to excise the chloramphenicol resistance cassette from the integrants in E.coli genome we transformed the cells with a plasmid (pCP20 plasmid) carrying the gene encoding for the flippase protein. This protein is able to cut fragment between two FRT flanking sequences. pCP20 was transformed in MC43-abc and MG43-abc and plated on LB+Amp agar plate.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 1st==<br />
<br />
This morning, we checked tranformed plates:<br />
<br />
*I72 showed NO colony!! :/ we will repeat this ligation next week<br />
*I70 and I71 showed colonies, 5 for each plate were picked and inoculated in 1ml LB+Amp to prepare glycerol stocks and for screening.<br />
*I73 showed colonies; six of them were picked and inoculated into 5 ml LB+Amp and let grow ON at 37°C, 220rpm in order to do E-P screeening the following day.<br />
<br />
----<br />
<br />
Transformation of I55 into ''E. coli'' DH5-alpha. It was plated on LB+Amp 100 agar plate and let grow ON at +37°C.<br />
<br />
----<br />
<br />
Miniprep and Nanodrop quantification for:<br />
*I31: 76,1 ng/ul<br />
*I35: 66,8 ng/ul<br />
*I60: 279,7 ng/ul<br />
(they were also sent sequencing)<br />
*INTEIN: 123,8 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-1: 104,8 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-2: 141,2 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-3: 113,5 ng/ul<br />
*I37: 174,9 ng/ul<br />
<br />
Digest:<br />
*<partinfo>pSB1C3</partinfo>: E-P<br />
*<partinfo>pSB1C3</partinfo>: E-S<br />
*<partinfo>pSB1C3</partinfo>: X-P<br />
*INTEIN: E-P<br />
*I60: E-S<br />
*I31: S-P<br />
*I35: E-P<br />
*I35: E-X<br />
*I37: E-X<br />
*I57: E-X<br />
<br />
Gel run/cut/extraction/quantification:<br />
{|align="center"<br />
|[[Image:UNIPV10_01_10_2010_digestioni_medio.jpg|thumb|200px|center|Intein, I31, I35, I37, I57, I60 digestions]] || [[Image:UNIPV10_01_10_2010_digestioni_psb1c3.jpg|thumb|150px|center|<partinfo>pSB1C3</partinfo> digested E-P (both vector and insert were kept), E-S, X-P.]]<br />
|}<br />
<br />
*I37 (E-X): 20,4 ng/ul<br />
*I57 (E-X): 17,6 ng/ul<br />
*I60 (E-S): 9,5 ng/ul<br />
They were stored at -20°C.<br />
*I31 (E-S): 5,4 ng/ul<br />
*I35 (E-P): 1,0 ng/ul ;-(<br />
*I35 (E-X): 14,4 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (E-P): 14,3 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (E-S): 15,2 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (X-P): 13,9 ng/ul<br />
<br />
ON ligation of:<br />
*I74: I31 (E-S) + I35 (E-X)<br />
*INTEIN_C3: INTEIN (E-P) + <partinfo>pSB1C3</partinfo> (E-P)<br />
<br />
<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 2nd==<br />
<br />
PCR from colony for I55 (eleven colonies were picked).<br />
<br />
Gel run:<br />
<br />
[[Image:UNIPV10_02_10_2010_I55_screening.jpg|thumb|200px|center|I55 colony PCR screening.]]<br />
<br />
We decided to make glycerol stock for I55-1. Inoculum into 5 ml LB+Amp of the positive culture that was let grow ON at 37°C, 220 rpm.<br />
----<br />
Miniprep and quantification for I73-1/2/3/4/5/6:<br />
*I73-1: 154 ng/ul<br />
*I73-2: 177 ng/ul<br />
*I73-3: 154,7 ng/ul<br />
*I73-4: 145,5ng/ul<br />
*I73-5: 152,6 ng/ul<br />
*I73-6: 154,2 ng/ul<br />
1,5 hours E-P digest (for screening) and gel run<br />
<br />
[[Image:UNIPV10_02_10_2010_I73_screening.jpg|thumb|200px|center|I70, I71, I73, A9_4C5, A99_4C5 screening.]]<br />
<br />
As you can see they are (except for I73-6) all positive. So we stocked I73-1 and stored it at -80°C.<br />
<br />
----<br />
Transformation of I74 and INTEIN_C3 (1ul) in ''E. coli'' TOP10. They were plated on LB+Amp100 and LB+Cm34 agar plates respectively. They were let grow ON at +37°C.<br />
<br />
----<br />
Inoculum of I55-1 into 5 ml LB+Amp. ON growth at +37°C, 220 rpm.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 3rd==<br />
Glycerol stock for:<br />
*I55-1<br />
*IC-1/2/3/4-4C5<br />
<br />
----<br />
Pick of a single non-red colony of INTEIN_C3 and inoculum into 5 ml LB+Cm34. Sample was let grow ON at +37°C, 220 rpm.<br />
<br />
----<br />
Miniprep and quantification of:<br />
*I55-1: 57,4 ng/ul<br />
*I55-1bis: 79,6 ng/ul<br />
<br />
I55-1bis was digested E-S for about three hours and than gel run/cut.<br />
<br />
----<br />
<br />
Colony PCR for I74-1..6.<br />
<br />
----<br />
Gel run for PCR samples and I55 (E-S) and cut for this one.<br />
[[Image:UNIPV10_03_10_2010_I74_screen_I55_cut.jpg|thumb|200px|center|I74 screening and I55 E-S cut.]]<br />
Very bad PCR. We performed a massive PCR from colony during the night (I74-7..24), we ran it the following day.<br />
<br />
Gel extraction and quantification:<br />
*I55 (E-S): 5,0 ng/ul<br />
<br />
Ligation of I55 (E-S) with already available DNA:<br />
*I75: I55 (E-S) + I37 (E-X)<br />
*I77: I55 (E-S) + I57 (E-X)<br />
----<br />
Inoculum of I35 (last quantification after gel extraction was too poor; we will digest it again E-P) and I54 into 5 ml LB+Amp: ON growth at +37°C, 220 rpm.<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
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</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/September/settimana5Team:UNIPV-Pavia/Calendar/September/settimana52010-10-20T17:09:45Z<p>Matteo: /* September, 30th */</p>
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<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
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{{UNIPV-Pavia/Style}}<br />
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<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
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<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana4|Week 4]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana5|Week 5]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>SEPTEMBER: WEEK 5</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==September, 27th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test27settembre|Tecan Test]]</font><br />
<br />
----<br />
<br />
MiniPrep and E-P digest for I62-a, I62-b, I62-c, I62-d, I62-e, I62-f<br />
<br />
DNA PCR for MyYeast 1-2-3<br />
<br />
Gel run<br />
[[Image:UNIPV10_27_09_2010_I62_screen.jpg|thumb|200px|center|I62 and MyYeast screening.]]<br />
<br />
<br />
I62-e was positive, so we made glycerol stock and stored it at -80°C.<br />
<br />
<br />
Protein electrophoresis for phasins.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 28th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test28settembre|Tecan Test]]</font><br />
<br />
----<br />
Miniprep and quantification for:<br />
*I1: 59,6 ng/ul<br />
*I4: 109,9 ng/ul<br />
*I53: 160,3 ng/ul<br />
*<partinfo>BBa_J04450</partinfo>-1:55,9 ng/ul<br />
*<partinfo>BBa_J04450</partinfo>-2: 47 ng/ul<br />
<br />
ON digestion of:<br />
*I1 (E-S)<br />
*I4 (E-S)<br />
*I53 (E-X)<br />
*<partinfo>BBa_J04450</partinfo>-1 (E-P)<br />
*<partinfo>BBa_J04450</partinfo>-2 (E-P)<br />
*MyYeast-3 (DNA already available) (E-S)<br />
*<partinfo>pSB4C5</partinfo> (DNA already available) (E-P)<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 29th==<br />
<br />
Colony PCR for 6 newly picked colonies.<br />
[[Image:UNIPV10_29_09_2010_I55_screen.jpg|thumb|200px|center|I55 colony PCR screening.]]<br />
No positives nor this time.<br />
----<br />
<br />
<br />
Gel run for all digested parts<br />
[[Image:UNIPV10_29_09_2010_digestions.jpg|thumb|200px|center|Digestions.]]<br />
and cut/gel purification for:<br />
*4C5 (E-P): 19,3 ng/ul<br />
*My Yeast 3 (E-S): 20,8 ng/ul<br />
*I1: 3,8 ng/ul<br />
*I4 (E-S): 14,2 ng/ul<br />
*I53 (E-X): 21,6 ng/ul<br />
*<partinfo>BBa_J04450</partinfo> (pLac-RBS-mRFP-TT) (E-P): 5,6 ng/ul<br />
*<partinfo>pSB1C3</partinfo>: 10 ng/ul<br />
<br />
<br />
Ligation of:<br />
{| align='center', border='1'<br />
| I70=MyYeast 3(E-S)+I4(E-S)<br />
|-<br />
| I71=MyYeast 3(E-S)+I1(E-S)<br />
|-<br />
|I72=<partinfo>BBa_J04450</partinfo>(E-P)+<partinfo>pSB4C5</partinfo><br />
|-<br />
|I73= I38 (E-S) + I53 (E-X)<br />
|}<br />
<br />
----<br />
<br />
Inoculum of MC43, MG43 in 5ml LB with chloramphenicol concentration of 12.5 ug/ml.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 30th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test16settembre|Tecan Test]]</font><br />
<br />
----<br />
<br />
Transformation of I73 (1ul) into ''E. coli'' TOP10. It was plated on LB+Amp 100 ug/ml agar plate.<br />
<br />
Trasformation of: <br />
*I70 (myYeast with I4, Ptef1-mOrange-tAdh1) in TOP10 and plated on LB+Amp<br />
*I71 (myYeast with I1, mOrange-tAdh1) in TOP10 and plated on LB+Amp<br />
*I72 (pLac-RFP in pSB4C5) in TOP10 and plated on LB+Cm 12,5<br />
<br />
All plates were incubated at 37°C ON<br />
<br />
<br />
Since I55 was negative again we decided to ligate it again (probably we had a bad digest for vector - I20 - and a good one for insert - we used I38 in other ligations without problems). We diegested already miniprepped DNA for three hours with 1 ul of enzymes:<br />
*I20: (E-X)<br />
<br />
Gel run/cut/extraction/quantification<br />
[[Image:UNIPV10_30_09_2010_I20_E_X.jpg|thumb|70px|center|I20 E-X digest.]]<br />
I20 E-X: 13,4 ng/ul<br />
<br />
We repeated I55 ligation using newly digested vector and already digested I38 (E-S)<br />
<br />
I55: I38 (E-S) + I20 (E-X)<br />
<br />
Inoculum into 5ml LB+Amp of:<br />
*I31<br />
*I35<br />
*I37<br />
*I57<br />
*I60<br />
*INTEIN<br />
*3x <partinfo>BBa_J04450</partinfo> (to take <partinfo>pSB1C3</partinfo>, so <partinfo>pSB1C3</partinfo> from now on)<br />
Cultures were let grow ON at 37°C, 220 rpm.<br />
<br />
----<br />
<br />
Inoculum of E. coli TOP10 in 1,5 ml LB, grown ON at 37°C 220 rpm. Tomorrow we will prepare competent cells!!<br />
<br />
Inoculum of:<br />
<br />
*S1 in 500ul M9+Cm 12,5<br />
*I47-S1 in 500ul M9+Amp+Cm 12,5<br />
*GFP-S1 in 500ul M9+Amp+Cm 12,5<br />
*RBS32 in 500ul M9+Amp<br />
<br />
Cultures were grown ON at 37°C 220 rpm.<br />
<br />
Tomorrow we will test the ability of these strains to produce GFP and to lyse when HSL is added.<br />
<br />
----<br />
First day of the flippase protocol. <br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 1st==<br />
<br />
This morning, we checked tranformed plates:<br />
<br />
*I72 showed NO colony!! :/ we will repeat this ligation next week<br />
*I70 and I71 showed colonies, 5 for each plate were picked and inoculated in 1ml LB+Amp to prepare glycerol stocks and for screening.<br />
*I73 showed colonies; six of them were picked and inoculated into 5 ml LB+Amp and let grow ON at 37°C, 220rpm in order to do E-P screeening the following day.<br />
<br />
----<br />
<br />
Transformation of I55 into ''E. coli'' DH5-alpha. It was plated on LB+Amp 100 agar plate and let grow ON at +37°C.<br />
<br />
----<br />
<br />
Miniprep and Nanodrop quantification for:<br />
*I31: 76,1 ng/ul<br />
*I35: 66,8 ng/ul<br />
*I60: 279,7 ng/ul<br />
(they were also sent sequencing)<br />
*INTEIN: 123,8 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-1: 104,8 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-2: 141,2 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-3: 113,5 ng/ul<br />
*I37: 174,9 ng/ul<br />
<br />
Digest:<br />
*<partinfo>pSB1C3</partinfo>: E-P<br />
*<partinfo>pSB1C3</partinfo>: E-S<br />
*<partinfo>pSB1C3</partinfo>: X-P<br />
*INTEIN: E-P<br />
*I60: E-S<br />
*I31: S-P<br />
*I35: E-P<br />
*I35: E-X<br />
*I37: E-X<br />
*I57: E-X<br />
<br />
Gel run/cut/extraction/quantification:<br />
{|align="center"<br />
|[[Image:UNIPV10_01_10_2010_digestioni_medio.jpg|thumb|200px|center|Intein, I31, I35, I37, I57, I60 digestions]] || [[Image:UNIPV10_01_10_2010_digestioni_psb1c3.jpg|thumb|150px|center|<partinfo>pSB1C3</partinfo> digested E-P (both vector and insert were kept), E-S, X-P.]]<br />
|}<br />
<br />
*I37 (E-X): 20,4 ng/ul<br />
*I57 (E-X): 17,6 ng/ul<br />
*I60 (E-S): 9,5 ng/ul<br />
They were stored at -20°C.<br />
*I31 (E-S): 5,4 ng/ul<br />
*I35 (E-P): 1,0 ng/ul ;-(<br />
*I35 (E-X): 14,4 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (E-P): 14,3 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (E-S): 15,2 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (X-P): 13,9 ng/ul<br />
<br />
ON ligation of:<br />
*I74: I31 (E-S) + I35 (E-X)<br />
*INTEIN_C3: INTEIN (E-P) + <partinfo>pSB1C3</partinfo> (E-P)<br />
<br />
<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 2nd==<br />
<br />
PCR from colony for I55 (eleven colonies were picked).<br />
<br />
Gel run:<br />
<br />
[[Image:UNIPV10_02_10_2010_I55_screening.jpg|thumb|200px|center|I55 colony PCR screening.]]<br />
<br />
We decided to make glycerol stock for I55-1. Inoculum into 5 ml LB+Amp of the positive culture that was let grow ON at 37°C, 220 rpm.<br />
----<br />
Miniprep and quantification for I73-1/2/3/4/5/6:<br />
*I73-1: 154 ng/ul<br />
*I73-2: 177 ng/ul<br />
*I73-3: 154,7 ng/ul<br />
*I73-4: 145,5ng/ul<br />
*I73-5: 152,6 ng/ul<br />
*I73-6: 154,2 ng/ul<br />
1,5 hours E-P digest (for screening) and gel run<br />
<br />
[[Image:UNIPV10_02_10_2010_I73_screening.jpg|thumb|200px|center|I70, I71, I73, A9_4C5, A99_4C5 screening.]]<br />
<br />
As you can see they are (except for I73-6) all positive. So we stocked I73-1 and stored it at -80°C.<br />
<br />
----<br />
Transformation of I74 and INTEIN_C3 (1ul) in ''E. coli'' TOP10. They were plated on LB+Amp100 and LB+Cm34 agar plates respectively. They were let grow ON at +37°C.<br />
<br />
----<br />
Inoculum of I55-1 into 5 ml LB+Amp. ON growth at +37°C, 220 rpm.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 3rd==<br />
Glycerol stock for:<br />
*I55-1<br />
*IC-1/2/3/4-4C5<br />
<br />
----<br />
Pick of a single non-red colony of INTEIN_C3 and inoculum into 5 ml LB+Cm34. Sample was let grow ON at +37°C, 220 rpm.<br />
<br />
----<br />
Miniprep and quantification of:<br />
*I55-1: 57,4 ng/ul<br />
*I55-1bis: 79,6 ng/ul<br />
<br />
I55-1bis was digested E-S for about three hours and than gel run/cut.<br />
<br />
----<br />
<br />
Colony PCR for I74-1..6.<br />
<br />
----<br />
Gel run for PCR samples and I55 (E-S) and cut for this one.<br />
[[Image:UNIPV10_03_10_2010_I74_screen_I55_cut.jpg|thumb|200px|center|I74 screening and I55 E-S cut.]]<br />
Very bad PCR. We performed a massive PCR from colony during the night (I74-7..24), we ran it the following day.<br />
<br />
Gel extraction and quantification:<br />
*I55 (E-S): 5,0 ng/ul<br />
<br />
Ligation of I55 (E-S) with already available DNA:<br />
*I75: I55 (E-S) + I37 (E-X)<br />
*I77: I55 (E-S) + I57 (E-X)<br />
----<br />
Inoculum of I35 (last quantification after gel extraction was too poor; we will digest it again E-P) and I54 into 5 ml LB+Amp: ON growth at +37°C, 220 rpm.<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
<!-- table previous next week --><br />
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<td align="left">[[Team:UNIPV-Pavia/Calendar/September/settimana4| Previous week]]</td><br />
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{{UNIPV-Pavia/menu_mesi}}<br />
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</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/September/settimana5Team:UNIPV-Pavia/Calendar/September/settimana52010-10-20T16:50:03Z<p>Matteo: /* September, 29th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
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{{UNIPV-Pavia/Style}}<br />
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<table class="menu" border="0" width="100%"><br />
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<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana4|Week 4]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana5|Week 5]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>SEPTEMBER: WEEK 5</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==September, 27th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test27settembre|Tecan Test]]</font><br />
<br />
----<br />
<br />
MiniPrep and E-P digest for I62-a, I62-b, I62-c, I62-d, I62-e, I62-f<br />
<br />
DNA PCR for MyYeast 1-2-3<br />
<br />
Gel run<br />
[[Image:UNIPV10_27_09_2010_I62_screen.jpg|thumb|200px|center|I62 and MyYeast screening.]]<br />
<br />
<br />
I62-e was positive, so we made glycerol stock and stored it at -80°C.<br />
<br />
<br />
Protein electrophoresis for phasins.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 28th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test28settembre|Tecan Test]]</font><br />
<br />
----<br />
Miniprep and quantification for:<br />
*I1: 59,6 ng/ul<br />
*I4: 109,9 ng/ul<br />
*I53: 160,3 ng/ul<br />
*<partinfo>BBa_J04450</partinfo>-1:55,9 ng/ul<br />
*<partinfo>BBa_J04450</partinfo>-2: 47 ng/ul<br />
<br />
ON digestion of:<br />
*I1 (E-S)<br />
*I4 (E-S)<br />
*I53 (E-X)<br />
*<partinfo>BBa_J04450</partinfo>-1 (E-P)<br />
*<partinfo>BBa_J04450</partinfo>-2 (E-P)<br />
*MyYeast-3 (DNA already available) (E-S)<br />
*<partinfo>pSB4C5</partinfo> (DNA already available) (E-P)<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 29th==<br />
<br />
Colony PCR for 6 newly picked colonies.<br />
[[Image:UNIPV10_29_09_2010_I55_screen.jpg|thumb|200px|center|I55 colony PCR screening.]]<br />
No positives nor this time.<br />
----<br />
<br />
<br />
Gel run for all digested parts<br />
[[Image:UNIPV10_29_09_2010_digestions.jpg|thumb|200px|center|Digestions.]]<br />
and cut/gel purification for:<br />
*4C5 (E-P): 19,3 ng/ul<br />
*My Yeast 3 (E-S): 20,8 ng/ul<br />
*I1: 3,8 ng/ul<br />
*I4 (E-S): 14,2 ng/ul<br />
*I53 (E-X): 21,6 ng/ul<br />
*<partinfo>BBa_J04450</partinfo> (pLac-RBS-mRFP-TT) (E-P): 5,6 ng/ul<br />
*<partinfo>pSB1C3</partinfo>: 10 ng/ul<br />
<br />
<br />
Ligation of:<br />
{| align='center', border='1'<br />
| I70=MyYeast 3(E-S)+I4(E-S)<br />
|-<br />
| I71=MyYeast 3(E-S)+I1(E-S)<br />
|-<br />
|I72=<partinfo>BBa_J04450</partinfo>(E-P)+<partinfo>pSB4C5</partinfo><br />
|-<br />
|I73= I38 (E-S) + I53 (E-X)<br />
|}<br />
<br />
----<br />
<br />
Inoculum of MC43, MG43 in 5ml LB with chloramphenicol concentration of 12.5 ug/ml.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 30th==<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test16settembre|Tecan Test]]</font><br />
<br />
----<br />
<br />
Transformation of I73 (1ul) into ''E. coli'' TOP10. It was plated on LB+Amp 100 ug/ml agar plate.<br />
<br />
Trasformation of: <br />
*I70 (myYeast with I4, Ptef1-mOrange-tAdh1) in TOP10 and plated on LB+Amp<br />
*I71 (myYeast with I1, mOrange-tAdh1) in TOP10 and plated on LB+Amp<br />
*I72 (pLac-RFP in pSB4C5) in TOP10 and plated on LB+Cm 12,5<br />
<br />
All plates were incubated at 37°C ON<br />
<br />
<br />
Since I55 was negative again we decided to ligate it again (probably we had a bad digest for vector - I20 - and a good one for insert - we used I38 in other ligations without problems). We diegested already miniprepped DNA for three hours with 1 ul of enzymes:<br />
*I20: (E-X)<br />
<br />
Gel run/cut/extraction/quantification<br />
[[Image:UNIPV10_30_09_2010_I20_E_X.jpg|thumb|70px|center|I20 E-X digest.]]<br />
I20 E-X: 13,4 ng/ul<br />
<br />
We repeated I55 ligation using newly digested vector and already digested I38 (E-S)<br />
<br />
I55: I38 (E-S) + I20 (E-X)<br />
<br />
Inoculum into 5ml LB+Amp of:<br />
*I31<br />
*I35<br />
*I37<br />
*I57<br />
*I60<br />
*INTEIN<br />
*3x <partinfo>BBa_J04450</partinfo> (to take <partinfo>pSB1C3</partinfo>, so <partinfo>pSB1C3</partinfo> from now on)<br />
Cultures were let grow ON at 37°C, 220 rpm.<br />
<br />
----<br />
<br />
Inoculum of E. coli TOP10 in 1,5 ml LB, grown ON at 37°C 220 rpm. Tomorrow we will prepare competent cells!!<br />
<br />
Inoculum of:<br />
<br />
*S1 in 500ul M9+Cm 12,5<br />
*I47-S1 in 500ul M9+Amp+Cm 12,5<br />
*GFP-S1 in 500ul M9+Amp+Cm 12,5<br />
*RBS32 in 500ul M9+Amp<br />
<br />
Cultures were grown ON at 37°C 220 rpm.<br />
<br />
Tomorrow we will test the ability of these strains to produce GFP and to lyse when HSL is added.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 1st==<br />
<br />
This morning, we checked tranformed plates:<br />
<br />
*I72 showed NO colony!! :/ we will repeat this ligation next week<br />
*I70 and I71 showed colonies, 5 for each plate were picked and inoculated in 1ml LB+Amp to prepare glycerol stocks and for screening.<br />
*I73 showed colonies; six of them were picked and inoculated into 5 ml LB+Amp and let grow ON at 37°C, 220rpm in order to do E-P screeening the following day.<br />
<br />
----<br />
<br />
Transformation of I55 into ''E. coli'' DH5-alpha. It was plated on LB+Amp 100 agar plate and let grow ON at +37°C.<br />
<br />
----<br />
<br />
Miniprep and Nanodrop quantification for:<br />
*I31: 76,1 ng/ul<br />
*I35: 66,8 ng/ul<br />
*I60: 279,7 ng/ul<br />
(they were also sent sequencing)<br />
*INTEIN: 123,8 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-1: 104,8 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-2: 141,2 ng/ul<br />
*<partinfo>pSB1C3</partinfo>-3: 113,5 ng/ul<br />
*I37: 174,9 ng/ul<br />
<br />
Digest:<br />
*<partinfo>pSB1C3</partinfo>: E-P<br />
*<partinfo>pSB1C3</partinfo>: E-S<br />
*<partinfo>pSB1C3</partinfo>: X-P<br />
*INTEIN: E-P<br />
*I60: E-S<br />
*I31: S-P<br />
*I35: E-P<br />
*I35: E-X<br />
*I37: E-X<br />
*I57: E-X<br />
<br />
Gel run/cut/extraction/quantification:<br />
{|align="center"<br />
|[[Image:UNIPV10_01_10_2010_digestioni_medio.jpg|thumb|200px|center|Intein, I31, I35, I37, I57, I60 digestions]] || [[Image:UNIPV10_01_10_2010_digestioni_psb1c3.jpg|thumb|150px|center|<partinfo>pSB1C3</partinfo> digested E-P (both vector and insert were kept), E-S, X-P.]]<br />
|}<br />
<br />
*I37 (E-X): 20,4 ng/ul<br />
*I57 (E-X): 17,6 ng/ul<br />
*I60 (E-S): 9,5 ng/ul<br />
They were stored at -20°C.<br />
*I31 (E-S): 5,4 ng/ul<br />
*I35 (E-P): 1,0 ng/ul ;-(<br />
*I35 (E-X): 14,4 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (E-P): 14,3 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (E-S): 15,2 ng/ul<br />
*<partinfo>pSB1C3</partinfo> (X-P): 13,9 ng/ul<br />
<br />
ON ligation of:<br />
*I74: I31 (E-S) + I35 (E-X)<br />
*INTEIN_C3: INTEIN (E-P) + <partinfo>pSB1C3</partinfo> (E-P)<br />
<br />
<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 2nd==<br />
<br />
PCR from colony for I55 (eleven colonies were picked).<br />
<br />
Gel run:<br />
<br />
[[Image:UNIPV10_02_10_2010_I55_screening.jpg|thumb|200px|center|I55 colony PCR screening.]]<br />
<br />
We decided to make glycerol stock for I55-1. Inoculum into 5 ml LB+Amp of the positive culture that was let grow ON at 37°C, 220 rpm.<br />
----<br />
Miniprep and quantification for I73-1/2/3/4/5/6:<br />
*I73-1: 154 ng/ul<br />
*I73-2: 177 ng/ul<br />
*I73-3: 154,7 ng/ul<br />
*I73-4: 145,5ng/ul<br />
*I73-5: 152,6 ng/ul<br />
*I73-6: 154,2 ng/ul<br />
1,5 hours E-P digest (for screening) and gel run<br />
<br />
[[Image:UNIPV10_02_10_2010_I73_screening.jpg|thumb|200px|center|I70, I71, I73, A9_4C5, A99_4C5 screening.]]<br />
<br />
As you can see they are (except for I73-6) all positive. So we stocked I73-1 and stored it at -80°C.<br />
<br />
----<br />
Transformation of I74 and INTEIN_C3 (1ul) in ''E. coli'' TOP10. They were plated on LB+Amp100 and LB+Cm34 agar plates respectively. They were let grow ON at +37°C.<br />
<br />
----<br />
Inoculum of I55-1 into 5 ml LB+Amp. ON growth at +37°C, 220 rpm.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 3rd==<br />
Glycerol stock for:<br />
*I55-1<br />
*IC-1/2/3/4-4C5<br />
<br />
----<br />
Pick of a single non-red colony of INTEIN_C3 and inoculum into 5 ml LB+Cm34. Sample was let grow ON at +37°C, 220 rpm.<br />
<br />
----<br />
Miniprep and quantification of:<br />
*I55-1: 57,4 ng/ul<br />
*I55-1bis: 79,6 ng/ul<br />
<br />
I55-1bis was digested E-S for about three hours and than gel run/cut.<br />
<br />
----<br />
<br />
Colony PCR for I74-1..6.<br />
<br />
----<br />
Gel run for PCR samples and I55 (E-S) and cut for this one.<br />
[[Image:UNIPV10_03_10_2010_I74_screen_I55_cut.jpg|thumb|200px|center|I74 screening and I55 E-S cut.]]<br />
Very bad PCR. We performed a massive PCR from colony during the night (I74-7..24), we ran it the following day.<br />
<br />
Gel extraction and quantification:<br />
*I55 (E-S): 5,0 ng/ul<br />
<br />
Ligation of I55 (E-S) with already available DNA:<br />
*I75: I55 (E-S) + I37 (E-X)<br />
*I77: I55 (E-S) + I57 (E-X)<br />
----<br />
Inoculum of I35 (last quantification after gel extraction was too poor; we will digest it again E-P) and I54 into 5 ml LB+Amp: ON growth at +37°C, 220 rpm.<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
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{{UNIPV-Pavia/menu_mesi}}<br />
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</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/September/settimana2Team:UNIPV-Pavia/Calendar/September/settimana22010-10-20T16:39:09Z<p>Matteo: /* September, 7th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
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<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana4|Week 4]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/September/settimana5|Week 5]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>SEPTEMBER: WEEK 2</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==September, 6th==<br />
Inoculum of I47, I48, I49 into 5 ml LB+Amp for TECAN experiment of the following day.<br />
<br />
----<br />
* Inoculum of I44-2 and I51-3 into 5 ml LB+Cm34 to evaluate MiniPrep yields.<br />
*Inoculum of I44-2 and I51-3 into 5 ml LB+Cm12,5 to evaluate MiniPrep yields and for sequencing.<br />
*Inoculum for MiniPrep and sequencing of:<br />
** <partinfo>BBa_J23100</partinfo>_4C5<br />
** <partinfo>BBa_J23101</partinfo>_4C5<br />
** <partinfo>BBa_J23105</partinfo>_4C5<br />
** <partinfo>BBa_J23106</partinfo>_4C5<br />
** <partinfo>BBa_J23110</partinfo>_4C5<br />
** <partinfo>BBa_J23114</partinfo>_4C5<br />
** <partinfo>BBa_J23116</partinfo>_4C5<br />
** <partinfo>BBa_J23118</partinfo>_4C5<br />
** I7-A<br />
** I8-5-D<br />
<br />
----<br />
Inoculum of cultures for TECAN test on self-inducible promoters:<br />
*I14-1<br />
*I15-1<br />
*I16-1<br />
*I17-1<br />
*I18-1<br />
*I19-1<br />
*I14_4C5-2<br />
*I15_4C5-2<br />
*I16_4C5-1<br />
*I17_4C5-1<br />
*I18_4C5-1<br />
*I19_4C5-1<br />
*I6-3<br />
*I3-1<br />
*<partinfo>BBa_B0031</partinfo><br />
<br />
----<br />
Glycerol stocks of MG43, MC42, MC43. Static TECAN test (absorbance, green fluorescence and red fluorescence) on 200 ul of these cultures.<br />
<br />
Cultures were diluted in 4 ml final of LB in order to obtain the same lowest OD with the following formula: culture to remove and substitute with LB=FinalVolume-(lowestOD/misuredOD)*FinalVolume.<br />
Centrifuge at 4000 rpm, 25°C for 5 min to pellet the culture, discard the surnatant and resuspend the pellet with 1 mL of M9. Static TECAN test (absorbance, green fluorescence and red fluorescence) on 200 ul of these cultures.<br />
<br />
----<br />
Liquid PCR was performed on MG43 (3 colonies), MC42 (3 colonies), MC43 (3 colonies), MC1061 (negative control) with the following parameters: T_annealing=52°C, cycles composed by 10 min at 94°C, 30 sec at 94°C, 30 sec at 52°C and 4 min at 72°C . This is the result:<br />
<br />
[[Image:UNIPV10_06_09_10_PCR_MG43ABC_MC42ABC_MC43ABC_Cneg_Blank.jpg|thumb|200px|center|PCR results for screening.]]<br />
<br />
----<br />
MiniPreps of I44-1:5 and I51-1:5 were performed with these results:<br />
<br />
{| border="1" align='center'<br />
| ''Culture'' || ''Quantifications (ng/ul)'' <br />
|-<br />
| I44-1 || 11,1<br />
|-<br />
| I44-2 || 9,9<br />
|-<br />
| I44-3 || 12,3<br />
|-<br />
| I44-4 || 11,4<br />
|-<br />
| I44-5 || 11,4<br />
|-<br />
| I51-1 || 13,6<br />
|-<br />
| I51-2 || 19,4<br />
|-<br />
| I51-3 || 16,5<br />
|-<br />
| I51-4 || 13,3<br />
|-<br />
| I51-5 || 21,8<br />
|}<br />
<br />
Probably was used the ethanol buffer without adding before the ethanol, during the wash step and so the quantifications were so low. <br />
<br />
Digestion of I44_1:5 and I51_1:5 performed as follow:<br />
<br />
{| border="1" align='center'<br />
| ''Culture'' || ''Kind'' || ''Final reaction volume (ul) '' || ''DNA (ul)'' || ''H20 (ul)'' || ''Enzyme 1 (ul)'' || ''Enzyme 2 (ul)'' || ''Buffer H (ul)'' <br />
|-<br />
| I44-1 || Screening || 25 || 21,5 || 0 || 0,5 EcoRI || 0,5 HindIII || 2,5<br />
|-<br />
| I44-2 || Screening || 25 || 21,5 || 0 || 0,5 EcoRI || 0,5 HindIII || 2,5<br />
|-<br />
| I44-3 || Screening || 25 || 21,5 || 0 || 0,5 EcoRI || 0,5 HindIII || 2,5<br />
|-<br />
| I44-4 || Screening || 25 || 21,5 || 0 || 0,5 EcoRI || 0,5 HindIII || 2,5<br />
|-<br />
| I44-5 || Screening || 25 || 21,5 || 0 || 0,5 EcoRI || 0,5 HindIII || 2,5<br />
|-<br />
| I51-1 || Screening || 25 || 21,5 || 0 || 0,5 EcoRI || 0,5 HindIII || 2,5<br />
|-<br />
| I51-2 || Screening || 25 || 21,5 || 0 || 0,5 EcoRI || 0,5 HindIII || 2,5<br />
|-<br />
| I51-3 || Screening || 25 || 21,5 || 0 || 0,5 EcoRI || 0,5 HindIII || 2,5<br />
|-<br />
| I51-4 || Screening || 25 || 21,5 || 0 || 0,5 EcoRI || 0,5 HindIII || 2,5<br />
|-<br />
| I51-5 || Screening || 25 || 21,5 || 0 || 0,5 EcoRI || 0,5 HindIII || 2,5<br />
|}<br />
<br />
Gel screening shows that all the constructs were ok, so we decided to choose I44-2 and I51-3.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 7th==<br />
<br />
MiniPrep for:<br />
<br />
{| border='1'<br />
|''' Culture'''||'''Quantifications (ng/ul)'''<br />
|-<br />
| <partinfo>BBa_J23100</partinfo>_4C5 || 11,8<br />
|-<br />
| <partinfo>BBa_J23101</partinfo>_4C5 || 39,7<br />
|-<br />
| <partinfo>BBa_J23105</partinfo>_4C5 || 25,4<br />
|-<br />
| <partinfo>BBa_J23106</partinfo>_4C5 || 29,7<br />
|-<br />
| <partinfo>BBa_J23110</partinfo>_4C5 || 24,7<br />
|-<br />
| <partinfo>BBa_J23114</partinfo>_4C5 || 15,4<br />
|-<br />
| <partinfo>BBa_J23116</partinfo>_4C5 || 20,7<br />
|-<br />
| <partinfo>BBa_J23118</partinfo>_4C5 || 20,4<br />
|-<br />
| I7-A || 113,6<br />
|-<br />
| I8-5-D || 127,6<br />
|-<br />
| I44-2 (Cm 34) || 63,9<br />
|-<br />
| I51-3 (Cm 34) || 100,4<br />
|-<br />
| I44-2 (Cm 12,5) || 68,1<br />
|-<br />
| I51-3 (Cm 12,5) || 135,4<br />
|-<br />
| I47 || 74,8<br />
|}<br />
<br />
----<br />
<br />
Cultures for TECAN test were diluted in the morning (h10:20) 1:100 (40ul in 4ml) and let grown at 37°C 220 rpm till they reach the desired OD.<br />
<br />
----<br />
Trasformation of I42 and I43 in MC008 and MG008 strains, strain DB3.1 without DNA and pSB1C3 RFP in DB3.1 strain.<br />
MG42, MC43, MC42, MC43 were plated on LB+Cm34 plates instead DB3.1 on LB+Cm12.5 plates.<br />
Integration protocol first day were performed.<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 8th==<br />
Inoculum of:<br />
*I31<br />
*I32<br />
*I35<br />
*I36<br />
*I37<br />
*I38<br />
*I40<br />
*I41<br />
*I20<br />
into 5 ml LB + Amp;<br />
already available:<br />
*I21(E-S)<br />
*I26(E-S)<br />
*I0(E-X)<br />
for second step of ligation of phasins/intein<br />
<br />
----<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 9th==<br />
Minipreps were quantified as follows:<br />
*I20: 113.5 ng/ul<br />
*I31: 76.4 ng/ul<br />
*I32: 85.4 ng/ul<br />
*I35: 77.2 ng/ul<br />
*I36: 83.8 ng/ul<br />
*I37: 156.5 ng/ul<br />
*I38: 94.4 ng/ul<br />
*I40: 556 ng/ul<br />
*I41: 148.2 ng/ul<br />
<br />
They were digested:<br />
*I20: SpeI-PstI<br />
*I31: EcoRI-XbaI<br />
*I32: E-S<br />
*I35: S-P<br />
*I36: E-S<br />
*I37: E-X; X-P<br />
*I38: E-S<br />
*I40: E-S<br />
*I41: E-X<br />
<br />
<br />
----<br />
MG42 and MG43 shown no colonies: probably the reason was a mistake on the choice of the MG008 strain tubes during the trasformations.<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 10th==<br />
<br />
Screening digestion of I44-2 and I51-3.<br />
<br />
{| border="1" align='center'<br />
| ''Culture'' || ''Kind'' || ''Final reaction volume (ul) '' || ''DNA (ul)'' || ''H20 (ul)'' || ''Enzyme 1 (ul)'' || ''Enzyme 2 (ul)'' || ''Buffer (ul)'' <br />
|-<br />
| I44-2 || Vector/Screening || 25 || 1,5 || 19 || 1 EcoRI || 1 PstII || 2,5 H<br />
|-<br />
| I44-2 || Vector/Screening || 25 || 1,5 || 19 || 1 EcoRI || 1 HindIII || 2,5 B<br />
|-<br />
| I44-2 || Vector/Screening || 25 || 1,5 || 19 || 1 NheI || 1 NheI || 2,5 T<br />
|-<br />
| I51-3 || Vector/Screening || 25 || 1 || 19,5 || 1 EcoRI || 1 PstII || 2,5 H<br />
|-<br />
| I51-3 || Vector/Screening || 25 || 1 || 19,5 || 1 EcoRI || 1 Hind || 2,5 B<br />
|-<br />
| I51-3 || Vector/Screening || 25 || 1 || 19,5 || 1 NheI || 1 NheI || 2,5 T<br />
|}<br />
<br />
[[Image:UNIPV10_10_09_10_Screening_I44(E-P)_I44(E-H)_I44(N-N)_I51(E-P)_I51(E-H)_I51(N-N).jpg|thumb|300px|center| Check for I44 and I51.]]<br />
<br />
Pipetted 5ul of the liquid cultures MC42, MC43 on LB+Cm12,5 and the culture spots were streaked on the plates. Plates were incubated ON at 43°C.<br />
<br />
----<br />
Gel run/cut for samples of previous day. Gel extraction showed the following quantifications:<br />
*I31 (E-X): 21.2 ng/ul<br />
*I32 (E-S): 11.3 ng/ul<br />
*I35 (S-P): 24.6 ng/ul<br />
*I36 (E-S): 12.1 ng/ul<br />
*I37 (E-X): 26 ng/ul<br />
*I37 (X-P): 12.2 ng/ul<br />
*I40 (E-S): 16.3 ng/ul<br />
*I41 (E-X): 24.4 ng/ul<br />
*I20 (S-P): 30.3 ng/ul<br />
<br />
Digestions were ligated in order to create:<br />
*I52: I35(S-P)+I37(X-P)<br />
*I53: I32(E-S)+I37(E-X)<br />
*I54: I36(E-S)+I37(E-X)<br />
*I55: I38(E-S)+I20(E-X)<br />
*I56: I26(E-S)+I41(E-X)<br />
*I57: I40(E-S)+I0(E-X)<br />
*I58: I38(E-S)+I31(E-X)<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==September, 11th==<br />
MC42 and MC43 plates were transfered at -4°C. <br />
<br />
I42 and I43 have been transformed in MG008 strain, plated on LB+Cm34 plates and incubated at 30°C for 48 hours.<br />
<br />
<br />
<br />
<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
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{{UNIPV-Pavia/menu_mesi}}<br />
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</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/October/settimana2Team:UNIPV-Pavia/Calendar/October/settimana22010-10-20T16:21:07Z<p>Matteo: /* October, 11th */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
<!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> <br />
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<br />
{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
<td valign="top"><br />
<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/October/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>OCTOBER: WEEK 2</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==October, 11th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
Inoculum of MC42-A, MC43-A, MG42-B, MG43-A, MC43_flip-a,b,c, MG43_flip-a,b,c, MC1061.<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 12th==<br />
<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 13th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 14th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 15th==<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==October, 16th==<br />
<br />
<br />
----<br />
<font color='red' size='+2'>We are now moving all our parts to <partinfo>pSB1C3</partinfo> plasmid</font><br />
----<br />
<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
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{{UNIPV-Pavia/menu_mesi}}<br />
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</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/August/settimana4Team:UNIPV-Pavia/Calendar/August/settimana42010-10-20T16:09:03Z<p>Matteo: /* August, 24th */</p>
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<div>__NOTOC__<br />
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[[Team:UNIPV-Pavia/Calendar/August/settimana1|Week 1]]<br />
</td><br />
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[[Team:UNIPV-Pavia/Calendar/August/settimana2|Week 2]]<br />
</td><br />
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[[Team:UNIPV-Pavia/Calendar/August/settimana3|Week 3]]<br />
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[[Team:UNIPV-Pavia/Calendar/August/settimana4|Week 4]]<br />
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<br />
<html><p align="center"><font size="4"><b>AUGUST: WEEK 4</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==August, 23rd==<br />
Colony PCR as screening for ligations I31..I41. For each plate three colonies were picked and PCR amplification was performed. At the same time colonies were let grow in 750 ml LB+Amp in order to be ready to make glycerol stocks for those with right amplicons.<br />
<br />
[[Image:UNIPV10_23_8_10_PCRscreeningI31_I41.jpg|thumb|300px|center|Colony PCR and gel run as screening for ligations I31..I41]]<br />
Only I31, I33, I34, I35, I37, I38, I41 are positive without any doubt (we took I31-1, I33-1. I34-1, I35-1, I37-1, I38-3, I41-1), while I39 were all wrong and we weren't sure about I32, I36 and I40 (we made however glycerol stocks for I32-2, I36-2, I40-1).<br />
We decided to repeat colony PCR for I32, I36, I39 and I40 next day.<br />
<br />
<br />
MyCrim-9, <partinfo>BBa_K173001</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>pSB4C5</partinfo> were digested E-P and MyCrim-9, Ring were digested HindIII for three hours.<br />
<br />
{| border="1" align='center'<br />
| ''Culture'' || ''Kind'' || ''Final reaction volume (ul) '' || ''DNA (ul)'' || ''H20 (ul)'' || ''Enzyme 1 (ul)'' || ''Enzyme 2 (ul)'' || ''Buffer (ul)'' <br />
|-<br />
| MyCrim || Vector || 25 || 6,5 || 14 || 1 EcoRI || 1 PstI || 2,5 H<br />
|-<br />
| MyCrim || Vector/Screening || 25 || 4 || 16,5 || 1 HindIII || 1 HindIII || 2,5 B<br />
|-<br />
| BBa_K173001 || Insert || 25 || 8 || 12,5 || 1 EcoRI || 1 PstI || 2,5 H<br />
|-<br />
| BBa_J23101 || Insert || 25 || 5 || 15,5 || 1 EcoRI || 1 PstI || 2,5 H<br />
|-<br />
| pSB4C5 || Insert || 25 || 5 || 15,5 || 1 EcoRI || 1 PstI || 2,5 H<br />
|-<br />
| Ring || Vector/Screening || 25 || 4 || 16,5 || 1 HindIII || 1 HindIII || 2,5 B<br />
|}<br />
<br />
Gel extraction of <partinfo>BBa_I52002</partinfo>(E-P) (insert of pSB4C5), MyCrim (E-P), BBa_K173001 (E-P), MyCrim (HindIII), vector of pSB4C5.<br />
Quantification: <br />
{| border="1" align='center'<br />
| ''Sample'' || ''Quantifications"<br />
|-<br />
| MyCrim (HindIII) || 13 ng/ul<br />
|-<br />
| MyCrim (E-P) || 23,4 ng/ul<br />
|-<br />
| BBa_K173001 (E-P) || 11 ng/ul<br />
|-<br />
| BBa_J23101 (E-P) || 9 ng/ul<br />
|-<br />
| pSB4C5 vector (E-P) || 23 ng/ul<br />
|-<br />
| BBa_I52002 (insert of pSB4C5) (E-P) || 6 ng/ul<br />
|}<br />
<br />
These parts were used to perform following ligations:<br />
<br />
{| border="1" align='center'<br />
| ''Name'' || ''Vector'' || ''Insert''<br />
|-<br />
| I42 || MyCrim (E-P) || BBa_K173001 (E-P)<br />
|-<br />
| I43 || MyCrim (E-P) || BBa_J23101 (E-P)<br />
|-<br />
| I44 || MyCrim (E-P) || BBa_I52002 (insert of pSB4C5) (E-P)<br />
|-<br />
| I45 || MyCrim (HindIII) || MyCrim (HindIII)<br />
|}<br />
<br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==August, 24th==<br />
Only this morning we remembered that I40 is in a commercial vector (pMA), we will screen it next day through an E-P digest (today we made glycerol stocks for other four colonies: I40-4/5/6/7) since it isn't possible with colony PCR.<br />
<br />
With this method we checked four new colonies of I32 (I32-4/5/6/7), I36 (I36-4/5/6/7) and I39 (I39/4/5/6/7).<br />
<br />
[[Image:UNIPV10_24_8_10_PCRscreening2_I32_I36_I39.jpg|thumb|300px|center|Colony PCR and gel run as screening for ligations I32, I36, I39]]<br />
<br />
I39-4/5 are right (we made glycerol stock for I39-5); I32 and I36 still show the presence of cotrasformation of our ligation and a simple phasin (we took I36-7 instead of I36-2: cleaner gel run). We inoculated 5ml LB+Amp with I40-1/4/5/6/7 and we let them grow ON at 37°C, 220 rpm for E-P digestion/screening.<br />
<br />
----<br />
<br />
Inoculum of:<br />
<br />
*<partinfo>BBa_J23105</partinfo><br />
*<partinfo>BBa_J23106</partinfo><br />
*<partinfo>BBa_J23114</partinfo><br />
*<partinfo>BBa_J23116</partinfo><br />
<br />
for tomorrow MiniPrep. They will be processed with other promoters, for wich we retrieved purified DNA from our freezer. These further parts are:<br />
<br />
*<partinfo>BBa_J23100</partinfo><br />
*<partinfo>BBa_J23101</partinfo> already digested E-P and purified<br />
*<partinfo>BBa_J23110</partinfo><br />
*<partinfo>BBa_J23118</partinfo><br />
*<partinfo>pSB4C5</partinfo> already digested E-P and purified<br />
<br />
These promoters, expressing RFP, will be moved from high copy plasmid <partinfo>pSB1A2</partinfo> to the low copy plasmid <partinfo>pSB4C5</partinfo> and will be tested in both condition in order to establish a strength ranking.<br />
<br />
----<br />
Inoculum of PBHR68 BioPlastic producing device and <partinfo>BBa_B0032</partinfo> from glycerol stock in 5ml LB+Amp to check the production of BioPlasic with Sudan Black staining protocol.<br />
<br />
Cultures were grown ON at 37°C, 220 rpm.<br />
<br />
----<br />
I42, I43 ligations were transformed in BW23474 strain and plated on selective LB agar plate with chloramphenicol concentration of 34 ug/ml. Instead I44 was transformed in DB3.1 strain and plated on the same type of plate of the other two transformations. <br />
<br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==August, 25th==<br />
Minipreps of I40-1/4/5/6/7 (to make the screening of ligations) were quantified as follows:<br />
*I40-1: 415,7 ng/ul<br />
*I40-4: 382,8 ng/ul<br />
*I40-5: 420 ng/ul<br />
*I40-6: 487,8 ng/ul<br />
*I40-7: 479,9 ng/ul<br />
<br />
Samples were digested E-P for three hours<br />
{| border="1" align='center'<br />
| ''Culture'' || ''Kind'' || ''Final reaction volume (ul) '' || ''DNA (ul)'' || ''H20 (ul)'' || ''Enzyme 1 (ul)'' || ''Enzyme 2 (ul)'' || ''Buffer H (ul)'' <br />
|-<br />
| I40-1 || Insert/Screening || 25 || 2 || 19,5 || 0,5 EcoRI || 0,5 PstI || 2,5<br />
|-<br />
| I40-4 || Insert/Screening || 25 || 2 || 19,5 || 0,5 EcoRI || 0,5 PstI || 2,5<br />
|-<br />
| I40-5 || Insert/Screening || 25 || 2 || 19,5 || 0,5 EcoRI || 0,5 PstI || 2,5<br />
|-<br />
| I40-6|| Insert/Screening || 25 || 2 || 19,5 || 0,5 EcoRI || 0,5 PstI || 2,5<br />
|-<br />
| I40-7 || Insert/Screening || 25 || 2 || 19,5 || 0,5 EcoRI || 0,5 PstI || 2,5<br />
|}<br />
and than gel run to check the length of ligations.<br />
[[Image:UNIPV10_25_8_10_E-PscreeningI40.jpg|thumb|300px|center|Gel run for I40-1/4/5/6/7 digested E-P]]<br />
As you can see all samples are positive, so we decided to keep glycerol stock of I40-1.<br />
<br />
----<br />
<br />
MiniPrep was performed for following cultures, and DNA was quantified as follows:<br />
{| align='center' border='1'<br />
| '''Culture name''' || '''Quantifiaction (ng/ul)'''<br />
|-<br />
| <partinfo>BBa_J23105</partinfo> || X ng/ul<br />
|-<br />
| <partinfo>BBa_J23106</partinfo> || X ng/ul<br />
|-<br />
| <partinfo>BBa_J23114</partinfo> || X ng/ul<br />
|-<br />
| <partinfo>BBa_J23116</partinfo> || X ng/ul<br />
|-||}<br />
<br />
Purified DNA was digested as follows:<br />
<br />
<br />
Digestion of:<br />
<br />
{| border="1" align='center'<br />
| ''Culture'' || ''Kind'' || ''Final reaction volume (ul) '' || ''DNA (ul)'' || ''H20 (ul)'' || ''Enzyme 1'' || ''Enzyme 2'' || ''Buffer H'' <br />
|-<br />
| <partinfo>BBa_J23100</partinfo> || Insert || 25 || 10 || 10,5 || 1 EcoRI || 1 PstI || 2,5<br />
|-<br />
| <partinfo>BBa_J23105</partinfo> || Insert || 25 || 10 || 10,5 || 1 EcoRI || 1 PstI || 2,5<br />
|-<br />
| <partinfo>BBa_J23106</partinfo> || Insert || 25 || 10 || 10,5 || 1 EcoRI || 1 PstI || 2,5<br />
|-<br />
| <partinfo>BBa_J23110</partinfo> || Insert || 25 || 10 || 10,5 || 1 EcoRI || 1 PstI || 2,5<br />
|-<br />
| <partinfo>BBa_J23114</partinfo> || Insert || 25 || 10 || 10,5 || 1 EcoRI || 1 PstI || 2,5<br />
|-<br />
| <partinfo>BBa_J23116</partinfo> || Insert || 25 || 10 || 10,5 || 1 EcoRI || 1 PstI || 2,5<br />
|-<br />
| <partinfo>BBa_J23118</partinfo> || Insert || 25 || 10 || 10,5 || 1 EcoRI || 1 PstI || 2,5<br />
<br />
|}<br />
<br />
Ligation of:<br />
<br />
* <partinfo>BBa_J23100</partinfo>_4C5=<partinfo>BBa_J23100</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>(E-P)<br />
* <partinfo>BBa_J23101</partinfo>_4C5=<partinfo>BBa_J23101</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>(E-P)<br />
* <partinfo>BBa_J23105</partinfo>_4C5=<partinfo>BBa_J23105</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>(E-P)<br />
* <partinfo>BBa_J23106</partinfo>_4C5=<partinfo>BBa_J23106</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>(E-P)<br />
* <partinfo>BBa_J23110</partinfo>_4C5=<partinfo>BBa_J23110</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>(E-P)<br />
* <partinfo>BBa_J23114</partinfo>_4C5=<partinfo>BBa_J23114</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>(E-P)<br />
* <partinfo>BBa_J23116</partinfo>_4C5=<partinfo>BBa_J23116</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>(E-P)<br />
* <partinfo>BBa_J23118</partinfo>_4C5=<partinfo>BBa_J23118</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>(E-P)<br />
<br />
----<br />
<br />
<br />
Preparation of samples for BioPlastic screening:<br />
<br />
* Cultures of PBHR68 and <partinfo>BBa_B0032</partinfo> were diluted 1:100 in fresh LB+Amp and werew prepared as follows:<br />
** <partinfo>BBa_B0032</partinfo> with NOTHING added (negative control)<br />
** PBHR68 with NOTHING added<br />
** PBHR68 + 2% glycerol (carbon source for BioPlastic production)<br />
** PBHR68 + 1mM IPTG (inducer for Plac promoter, expressing BioPlastic enzymes)<br />
** PBHR68 + 2% glycerol + 1mM IPTG<br />
<br />
After 8 hours, Sudan Black staining protocol was performed on 70ul cultures and 5 microscope slides were prepared. The resulting images are shown here:<br />
<br />
{|align="center"<br />
|-<br />
|[[Image:UNIPV10_PBHR68_nothing.jpg|thumb|200px|center|PBHR68 with nothing added in the culture, after 8 hours]] || |[[Image:UNIPV10_PBHR68_nothing_2.jpg|thumb|200px|center|PBHR68 with nothing added in the culture, after 8 hours]]<br />
|-<br />
|[[Image:UNIPV10_RBS32_nothing.jpg|thumb|200px|center|<partinfo>BBa_B0032</partinfo> with nothing added in the culture, after 8 hours (negative control)]] || |[[Image:UNIPV10_RBS32_nothing_2.jpg|thumb|200px|center|<partinfo>BBa_B0032</partinfo> with nothing added in the culture, after 8 hours (negative control)]] <br />
|-<br />
|[[Image:UNIPV10_PBHR68_gly.jpg|thumb|200px|center|PBHR68 with 2% glycerol added in the culture, after 8 hours]] || |[[Image:UNIPV10_PBHR68_gly_2.jpg|thumb|200px|center|PBHR68 with 2% glycerol added in the culture, after 8 hours]]<br />
|-<br />
|[[Image:UNIPV10_PBHR68_IPTG.jpg|thumb|200px|center|PBHR68 with 1mM IPTG added in the culture, after 8 hours]] || |[[Image:UNIPV10_PBHR68_IPTG_2.jpg|thumb|200px|center|PBHR68 with 1mM IPTG in the culture, after 8 hours]]<br />
|-<br />
|[[Image:UNIPV10_PBHR68_gly_IPTG.jpg|thumb|200px|center|PBHR68 with 1mM IPTG and 2% glycerol added in the culture, after 8 hours]] || |[[Image:UNIPV10_PBHR68_gly_IPTG_2.jpg|thumb|200px|center|PBHR68 with 1mM IPTG and 2% glycerol added in the culture, after 8 hours]]<br />
|}<br />
<br />
Cultures were further incubated for 22 hours at 37°C, 220 rpm. Tomorrow, we will repeat the staining protocol after 30 hours from inoculum to check the time ''E. coli'' takes to produce BioPlastic granules.<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==August, 26th==<br />
<br />
Transformation of ligations:<br />
<br />
{| border='1' align='center'<br />
|'''Ligation name''' || '''Strain''' || '''Resistance'''<br />
|-<br />
|<partinfo>BBa_J23100</partinfo>_4C5 || TOP10 || Cm 12,5<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>_4C5 || TOP10 || Cm 12,5<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>_4C5 || TOP10 || Cm 12,5<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>_4C5 || TOP10 || Cm 12,5<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>_4C5 || TOP10 || Cm 12,5<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>_4C5 || TOP10 || Cm 12,5<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>_4C5 || TOP10 || Cm 12,5<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>_4C5 || TOP10 || Cm 12,5<br />
|}<br />
<br />
Plates were incubated at 37°C overnight.<br />
<br />
Inoculum of self inducible promoters for TECAN test:<br />
<br />
{| border='1'<br />
| I7 ||I8 || I9 || I10<br />
|-<br />
| I12 || I8_4C5 || I15 || I16<br />
|-<br />
| I17 || I18|| I19 || I27<br />
|-<br />
| I28 || I30|| <partinfo>BBa_B0032</partinfo> || A2 (<partinfo>BBa_J23101</partinfo>+GFP)<br />
|}<br />
<br />
----<br />
After 30 hours, Sudan Black staining protocol was performed on 70ul cultures and 5 microscope slides were prepared. The resulting images are shown here:<br />
<br />
{|align="center"<br />
|-<br />
|[[Image:UNIPV10_PBHR68_nothing_30.jpg|thumb|200px|center|PBHR68 with nothing added in the culture, after 30 hours]] || |[[Image:UNIPV10_PBHR68_nothing_2_30.jpg|thumb|200px|center|PBHR68 with nothing added in the culture, after 30 hours]]<br />
|-<br />
|[[Image:UNIPV10_RBS32_nothing_30.jpg|thumb|200px|center|<partinfo>BBa_B0032</partinfo> with nothing added in the culture, after 30 hours (negative control)]] || |[[Image:UNIPV10_RBS32_nothing_2_30.jpg|thumb|200px|center|<partinfo>BBa_B0032</partinfo> with nothing added in the culture, after 30 hours (negative control)]] <br />
|-<br />
|[[Image:UNIPV10_PBHR68_gly_30.jpg|thumb|200px|center|PBHR68 with 2% glycerol added in the culture, after 30 hours]] || |[[Image:UNIPV10_PBHR68_gly_2_30.jpg|thumb|200px|center|PBHR68 with 2% glycerol added in the culture, after 30 hours]]<br />
|-<br />
|[[Image:UNIPV10_PBHR68_IPTG_30.jpg|thumb|200px|center|PBHR68 with 1mM IPTG added in the culture, after 30 hours]] || |[[Image:UNIPV10_PBHR68_IPTG_2_30.jpg|thumb|200px|center|PBHR68 with 1mM IPTG in the culture, after 30 hours]]<br />
|-<br />
|[[Image:UNIPV10_PBHR68_gly_IPTG_30.jpg|thumb|200px|center|PBHR68 with 1mM IPTG and 2% glycerol added in the culture, after 30 hours]] || |[[Image:UNIPV10_PBHR68_gly_IPTG_2_30.jpg|thumb|200px|center|PBHR68 with 1mM IPTG and 2% glycerol added in the culture, after 30 hours]]<br />
|}<br />
<br />
----<br />
Inoculum of<br />
*<partinfo>BBa_J13002</partinfo><br />
*I33<br />
*I34<br />
*I39<br />
*I41<br />
in 5 ml LB+Amp<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==August, 27th==<br />
Previously inoculated strains were miniprepped and quantified as follows:<br />
*<partinfo>BBa_J13002</partinfo>: 29,9 ng/ul<br />
*I33: 171,6 ng/ul<br />
*I34: 219,4 ng/ul<br />
*I39: 21,2 ng/ul<br />
*I41: 94,7 ng/ul<br />
<br />
Digestions (3,5 hours) for ligations:<br />
*I47: <partinfo>BBa_J13002</partinfo> (S-P) + I33 (X-P)<br />
*I48: I39 (S-P) + I34 (X-P)<br />
*I49: I39 (S-P) + I41 (X-P)<br />
<br />
{| border="1" align='center'<br />
| ''Culture'' || ''Kind'' || ''Final reaction volume (ul) '' || ''DNA (ul)'' || ''H20 (ul)'' || ''Enzyme 1 (ul)'' || ''Enzyme 2 (ul)'' || ''Buffer H (ul)'' <br />
|-<br />
| <partinfo>BBa_J13002</partinfo> || Vector || 25 || 21,5 || 0 || 0,5 SpeI || 0,5 PstI || 2,5<br />
|-<br />
| I39 || Vector || 25 || 21,5 || 0 || 0,5 S || 0,5 P || 2,5<br />
|-<br />
| I33 || Insert || 25 || 10,5 || 11 || 0,5 XbaI || 0,5 P || 2,5<br />
|-<br />
| I34 || Insert|| 25 || 13,5 || 8 || 0,5 X || 0,5 P || 2,5<br />
|-<br />
| I41 || Insert || 25 || 2 || 19,5 || 0,5 X || 0,5 P || 2,5<br />
|}<br />
<br />
Samples were loaded into a medium gel, run and cut<br />
[[Image:UNIPV10_27_8_10_digest_step2A.jpg|thumb|300px|center|Samples gel run/cut]]<br />
<br />
Gel extraction was quantified as follows:<br />
*<partinfo>BBa_J13002</partinfo> (S-P): 19,9 ng/ul<br />
*I33 (X-P): 17,7 ng/ul<br />
*I34 (X-P): 11,1 ng/ul<br />
*I39 (S-P): 4,7 ng/ul<br />
*I41 (X-P): 6,2 ng/ul<br />
<br />
Ligation of<br />
*I47: <partinfo>BBa_J13002</partinfo> (S-P) + I33 (X-P)<br />
*I48: I39 (S-P) + I34 (X-P)<br />
*I49: I39 (S-P) + I41 (X-P)<br />
<br />
<br />
----<br />
<br />
Plates containing the transformed ligations were all grown. Colony PCR was performed to screen if the length of insert was correct.<br />
<br />
{| align='center' border='1'<br />
| '''ligation name'''|| '''number of colonies screened'''<br />
|-<br />
| <partinfo>BBa_J23100</partinfo>_4C5 || 2 colonies<br />
|-<br />
| <partinfo>BBa_J23101</partinfo>_4C5 || 2 colonies<br />
|-<br />
| <partinfo>BBa_J23105</partinfo>_4C5 || 2 colonies<br />
|-<br />
| <partinfo>BBa_J23106</partinfo>_4C5 || 2 colonies<br />
|-<br />
| <partinfo>BBa_J23110</partinfo>_4C5 || 2 colonies<br />
|-<br />
| <partinfo>BBa_J23114</partinfo>_4C5 || 1 colony<br />
|-<br />
| <partinfo>BBa_J23116</partinfo>_4C5 || 2 colonies<br />
|-<br />
| <partinfo>BBa_J23118</partinfo>_4C5 || 2 colonies<br />
|}<br />
<br />
<br />
[[Image:UNIPV10_screening_promoters_Anserson_low_copy.jpg|thumb|300px|center|PCR screening for Anderson Promoters transferred in low copy plasmid <partinfo>pSB4C5</partinfo>]]<br />
<br />
Gel results show that we have the correct clones for <partinfo>BBa_J23100</partinfo>_4C5, <partinfo>BBa_J23101</partinfo>_4C5, <partinfo>BBa_J23105</partinfo>_4C5, <partinfo>BBa_J23106</partinfo>_4C5, <partinfo>BBa_J23110</partinfo>_4C5, <partinfo>BBa_J23114</partinfo>_4C5. Glycerol stocks were prepared for these parts and are stored at -80°C. <br />
<br />
<partinfo>BBa_J23116</partinfo>_4C5 and <partinfo>BBa_J23118</partinfo>_4C5 need to be further screened, so we will pick other colonies next week and we will perform a new colony PCR.<br />
----<br />
<br />
<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test27agosto|Tecan Test]]</font> was performed on prepared samples, after the usual protocol (dilution, medium change and dilution 1:1000).<br />
<br />
----<br />
Transformation of I29 in ''E. coli'' TOP10<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==August, 28th==<br />
Ligations I47, I48, I49 were stored at +4°C.<br />
<br />
----<br />
I29 plate was stored at +4°C.<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
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{{UNIPV-Pavia/menu_mesi}}<br />
<br />
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</table></div>Matteohttp://2010.igem.org/Team:UNIPV-Pavia/Calendar/August/settimana4Team:UNIPV-Pavia/Calendar/August/settimana42010-10-20T16:00:40Z<p>Matteo: /* August, 23rd */</p>
<hr />
<div>__NOTOC__<br />
<table width="100%" border="0"><br />
<tr><br />
<td colspan="2"> {{UNIPV-Pavia/header}} </td> <br />
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{{UNIPV-Pavia/Style}}<br />
<br />
<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><br />
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<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"><br />
<table class="menu" border="0" width="100%"><br />
<tr><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/August/settimana1|Week 1]]<br />
</td><br />
<td align="center" style="padding:0; height:20px"><br />
[[Team:UNIPV-Pavia/Calendar/August/settimana2|Week 2]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/August/settimana3|Week 3]]<br />
</td><br />
<td align="center"><br />
[[Team:UNIPV-Pavia/Calendar/August/settimana4|Week 4]]<br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<html><p align="center"><font size="4"><b>AUGUST: WEEK 4</b></font></p></html><hr><br><br />
<html><a name="indice"/></html><br />
<br />
==August, 23rd==<br />
Colony PCR as screening for ligations I31..I41. For each plate three colonies were picked and PCR amplification was performed. At the same time colonies were let grow in 750 ml LB+Amp in order to be ready to make glycerol stocks for those with right amplicons.<br />
<br />
[[Image:UNIPV10_23_8_10_PCRscreeningI31_I41.jpg|thumb|300px|center|Colony PCR and gel run as screening for ligations I31..I41]]<br />
Only I31, I33, I34, I35, I37, I38, I41 are positive without any doubt (we took I31-1, I33-1. I34-1, I35-1, I37-1, I38-3, I41-1), while I39 were all wrong and we weren't sure about I32, I36 and I40 (we made however glycerol stocks for I32-2, I36-2, I40-1).<br />
We decided to repeat colony PCR for I32, I36, I39 and I40 next day.<br />
<br />
<br />
MyCrim-9, <partinfo>BBa_K173001</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>pSB4C5</partinfo> were digested E-P and MyCrim-9, Ring were digested HindIII for three hours.<br />
<br />
{| border="1" align='center'<br />
| ''Culture'' || ''Kind'' || ''Final reaction volume (ul) '' || ''DNA (ul)'' || ''H20 (ul)'' || ''Enzyme 1 (ul)'' || ''Enzyme 2 (ul)'' || ''Buffer (ul)'' <br />
|-<br />
| MyCrim || Vector || 25 || 6,5 || 14 || 1 EcoRI || 1 PstI || 2,5 H<br />
|-<br />
| MyCrim || Vector/Screening || 25 || 4 || 16,5 || 1 HindIII || 1 HindIII || 2,5 B<br />
|-<br />
| BBa_K173001 || Insert || 25 || 8 || 12,5 || 1 EcoRI || 1 PstI || 2,5 H<br />
|-<br />
| BBa_J23101 || Insert || 25 || 5 || 15,5 || 1 EcoRI || 1 PstI || 2,5 H<br />
|-<br />
| pSB4C5 || Insert || 25 || 5 || 15,5 || 1 EcoRI || 1 PstI || 2,5 H<br />
|-<br />
| Ring || Vector/Screening || 25 || 4 || 16,5 || 1 HindIII || 1 HindIII || 2,5 B<br />
|}<br />
<br />
Gel extraction of <partinfo>BBa_I52002</partinfo>(E-P) (insert of pSB4C5), MyCrim (E-P), BBa_K173001 (E-P), MyCrim (HindIII), vector of pSB4C5.<br />
Quantification: <br />
{| border="1" align='center'<br />
| ''Sample'' || ''Quantifications"<br />
|-<br />
| MyCrim (HindIII) || 13 ng/ul<br />
|-<br />
| MyCrim (E-P) || 23,4 ng/ul<br />
|-<br />
| BBa_K173001 (E-P) || 11 ng/ul<br />
|-<br />
| BBa_J23101 (E-P) || 9 ng/ul<br />
|-<br />
| pSB4C5 vector (E-P) || 23 ng/ul<br />
|-<br />
| BBa_I52002 (insert of pSB4C5) (E-P) || 6 ng/ul<br />
|}<br />
<br />
These parts were used to perform following ligations:<br />
<br />
{| border="1" align='center'<br />
| ''Name'' || ''Vector'' || ''Insert''<br />
|-<br />
| I42 || MyCrim (E-P) || BBa_K173001 (E-P)<br />
|-<br />
| I43 || MyCrim (E-P) || BBa_J23101 (E-P)<br />
|-<br />
| I44 || MyCrim (E-P) || BBa_I52002 (insert of pSB4C5) (E-P)<br />
|-<br />
| I45 || MyCrim (HindIII) || MyCrim (HindIII)<br />
|}<br />
<br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==August, 24th==<br />
Only this morning we remembered that I40 is in a commercial vector (pMA), we will screen it next day through an E-P digest (today we made glycerol stocks for other four colonies: I40-4/5/6/7) since it isn't possible with colony PCR.<br />
<br />
With this method we checked four new colonies of I32 (I32-4/5/6/7), I36 (I36-4/5/6/7) and I39 (I39/4/5/6/7).<br />
<br />
[[Image:UNIPV10_24_8_10_PCRscreening2_I32_I36_I39.jpg|thumb|300px|center|Colony PCR and gel run as screening for ligations I32, I36, I39]]<br />
<br />
I39-4/5 are right (we made glycerol stock for I39-5); I32 and I36 still show the presence of cotrasformation of our ligation and a simple phasin (we took I36-7 instead of I36-2: cleaner gel run). We inoculated 5ml LB+Amp with I40-1/4/5/6/7 and we let them grow ON at 37°C, 220 rpm for E-P digestion/screening.<br />
<br />
----<br />
<br />
Inoculum of:<br />
<br />
*<partinfo>BBa_J23105</partinfo><br />
*<partinfo>BBa_J23106</partinfo><br />
*<partinfo>BBa_J23114</partinfo><br />
*<partinfo>BBa_J23116</partinfo><br />
<br />
for tomorrow MiniPrep. They will be processed with other promoters, for wich we retrieved purified DNA from our freezer. These further parts are:<br />
<br />
*<partinfo>BBa_J23100</partinfo><br />
*<partinfo>BBa_J23101</partinfo> already digested E-P and purified<br />
*<partinfo>BBa_J23110</partinfo><br />
*<partinfo>BBa_J23118</partinfo><br />
*<partinfo>pSB4C5</partinfo> already digested E-P and purified<br />
<br />
These promoters, expressing RFP, will be moved from high copy plasmid <partinfo>pSB1A2</partinfo> to the low copy plasmid <partinfo>pSB4C5</partinfo> and will be tested in both condition in order to establish a strength ranking.<br />
<br />
----<br />
Inoculum of PBHR68 BioPlastic producing device and <partinfo>BBa_B0032</partinfo> from glycerol stock in 5ml LB+Amp to check the production of BioPlasic with Sudan Black staining protocol.<br />
<br />
Cultures were grown ON at 37°C, 220 rpm.<br />
<br />
<br />
----<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==August, 25th==<br />
Minipreps of I40-1/4/5/6/7 (to make the screening of ligations) were quantified as follows:<br />
*I40-1: 415,7 ng/ul<br />
*I40-4: 382,8 ng/ul<br />
*I40-5: 420 ng/ul<br />
*I40-6: 487,8 ng/ul<br />
*I40-7: 479,9 ng/ul<br />
<br />
Samples were digested E-P for three hours<br />
{| border="1" align='center'<br />
| ''Culture'' || ''Kind'' || ''Final reaction volume (ul) '' || ''DNA (ul)'' || ''H20 (ul)'' || ''Enzyme 1 (ul)'' || ''Enzyme 2 (ul)'' || ''Buffer H (ul)'' <br />
|-<br />
| I40-1 || Insert/Screening || 25 || 2 || 19,5 || 0,5 EcoRI || 0,5 PstI || 2,5<br />
|-<br />
| I40-4 || Insert/Screening || 25 || 2 || 19,5 || 0,5 EcoRI || 0,5 PstI || 2,5<br />
|-<br />
| I40-5 || Insert/Screening || 25 || 2 || 19,5 || 0,5 EcoRI || 0,5 PstI || 2,5<br />
|-<br />
| I40-6|| Insert/Screening || 25 || 2 || 19,5 || 0,5 EcoRI || 0,5 PstI || 2,5<br />
|-<br />
| I40-7 || Insert/Screening || 25 || 2 || 19,5 || 0,5 EcoRI || 0,5 PstI || 2,5<br />
|}<br />
and than gel run to check the length of ligations.<br />
[[Image:UNIPV10_25_8_10_E-PscreeningI40.jpg|thumb|300px|center|Gel run for I40-1/4/5/6/7 digested E-P]]<br />
As you can see all samples are positive, so we decided to keep glycerol stock of I40-1.<br />
<br />
----<br />
<br />
MiniPrep was performed for following cultures, and DNA was quantified as follows:<br />
{| align='center' border='1'<br />
| '''Culture name''' || '''Quantifiaction (ng/ul)'''<br />
|-<br />
| <partinfo>BBa_J23105</partinfo> || X ng/ul<br />
|-<br />
| <partinfo>BBa_J23106</partinfo> || X ng/ul<br />
|-<br />
| <partinfo>BBa_J23114</partinfo> || X ng/ul<br />
|-<br />
| <partinfo>BBa_J23116</partinfo> || X ng/ul<br />
|-||}<br />
<br />
Purified DNA was digested as follows:<br />
<br />
<br />
Digestion of:<br />
<br />
{| border="1" align='center'<br />
| ''Culture'' || ''Kind'' || ''Final reaction volume (ul) '' || ''DNA (ul)'' || ''H20 (ul)'' || ''Enzyme 1'' || ''Enzyme 2'' || ''Buffer H'' <br />
|-<br />
| <partinfo>BBa_J23100</partinfo> || Insert || 25 || 10 || 10,5 || 1 EcoRI || 1 PstI || 2,5<br />
|-<br />
| <partinfo>BBa_J23105</partinfo> || Insert || 25 || 10 || 10,5 || 1 EcoRI || 1 PstI || 2,5<br />
|-<br />
| <partinfo>BBa_J23106</partinfo> || Insert || 25 || 10 || 10,5 || 1 EcoRI || 1 PstI || 2,5<br />
|-<br />
| <partinfo>BBa_J23110</partinfo> || Insert || 25 || 10 || 10,5 || 1 EcoRI || 1 PstI || 2,5<br />
|-<br />
| <partinfo>BBa_J23114</partinfo> || Insert || 25 || 10 || 10,5 || 1 EcoRI || 1 PstI || 2,5<br />
|-<br />
| <partinfo>BBa_J23116</partinfo> || Insert || 25 || 10 || 10,5 || 1 EcoRI || 1 PstI || 2,5<br />
|-<br />
| <partinfo>BBa_J23118</partinfo> || Insert || 25 || 10 || 10,5 || 1 EcoRI || 1 PstI || 2,5<br />
<br />
|}<br />
<br />
Ligation of:<br />
<br />
* <partinfo>BBa_J23100</partinfo>_4C5=<partinfo>BBa_J23100</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>(E-P)<br />
* <partinfo>BBa_J23101</partinfo>_4C5=<partinfo>BBa_J23101</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>(E-P)<br />
* <partinfo>BBa_J23105</partinfo>_4C5=<partinfo>BBa_J23105</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>(E-P)<br />
* <partinfo>BBa_J23106</partinfo>_4C5=<partinfo>BBa_J23106</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>(E-P)<br />
* <partinfo>BBa_J23110</partinfo>_4C5=<partinfo>BBa_J23110</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>(E-P)<br />
* <partinfo>BBa_J23114</partinfo>_4C5=<partinfo>BBa_J23114</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>(E-P)<br />
* <partinfo>BBa_J23116</partinfo>_4C5=<partinfo>BBa_J23116</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>(E-P)<br />
* <partinfo>BBa_J23118</partinfo>_4C5=<partinfo>BBa_J23118</partinfo>(E-P)+<partinfo>pSB4C5</partinfo>(E-P)<br />
<br />
----<br />
<br />
<br />
Preparation of samples for BioPlastic screening:<br />
<br />
* Cultures of PBHR68 and <partinfo>BBa_B0032</partinfo> were diluted 1:100 in fresh LB+Amp and werew prepared as follows:<br />
** <partinfo>BBa_B0032</partinfo> with NOTHING added (negative control)<br />
** PBHR68 with NOTHING added<br />
** PBHR68 + 2% glycerol (carbon source for BioPlastic production)<br />
** PBHR68 + 1mM IPTG (inducer for Plac promoter, expressing BioPlastic enzymes)<br />
** PBHR68 + 2% glycerol + 1mM IPTG<br />
<br />
After 8 hours, Sudan Black staining protocol was performed on 70ul cultures and 5 microscope slides were prepared. The resulting images are shown here:<br />
<br />
{|align="center"<br />
|-<br />
|[[Image:UNIPV10_PBHR68_nothing.jpg|thumb|200px|center|PBHR68 with nothing added in the culture, after 8 hours]] || |[[Image:UNIPV10_PBHR68_nothing_2.jpg|thumb|200px|center|PBHR68 with nothing added in the culture, after 8 hours]]<br />
|-<br />
|[[Image:UNIPV10_RBS32_nothing.jpg|thumb|200px|center|<partinfo>BBa_B0032</partinfo> with nothing added in the culture, after 8 hours (negative control)]] || |[[Image:UNIPV10_RBS32_nothing_2.jpg|thumb|200px|center|<partinfo>BBa_B0032</partinfo> with nothing added in the culture, after 8 hours (negative control)]] <br />
|-<br />
|[[Image:UNIPV10_PBHR68_gly.jpg|thumb|200px|center|PBHR68 with 2% glycerol added in the culture, after 8 hours]] || |[[Image:UNIPV10_PBHR68_gly_2.jpg|thumb|200px|center|PBHR68 with 2% glycerol added in the culture, after 8 hours]]<br />
|-<br />
|[[Image:UNIPV10_PBHR68_IPTG.jpg|thumb|200px|center|PBHR68 with 1mM IPTG added in the culture, after 8 hours]] || |[[Image:UNIPV10_PBHR68_IPTG_2.jpg|thumb|200px|center|PBHR68 with 1mM IPTG in the culture, after 8 hours]]<br />
|-<br />
|[[Image:UNIPV10_PBHR68_gly_IPTG.jpg|thumb|200px|center|PBHR68 with 1mM IPTG and 2% glycerol added in the culture, after 8 hours]] || |[[Image:UNIPV10_PBHR68_gly_IPTG_2.jpg|thumb|200px|center|PBHR68 with 1mM IPTG and 2% glycerol added in the culture, after 8 hours]]<br />
|}<br />
<br />
Cultures were further incubated for 22 hours at 37°C, 220 rpm. Tomorrow, we will repeat the staining protocol after 30 hours from inoculum to check the time ''E. coli'' takes to produce BioPlastic granules.<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==August, 26th==<br />
<br />
Transformation of ligations:<br />
<br />
{| border='1' align='center'<br />
|'''Ligation name''' || '''Strain''' || '''Resistance'''<br />
|-<br />
|<partinfo>BBa_J23100</partinfo>_4C5 || TOP10 || Cm 12,5<br />
|-<br />
|<partinfo>BBa_J23101</partinfo>_4C5 || TOP10 || Cm 12,5<br />
|-<br />
|<partinfo>BBa_J23105</partinfo>_4C5 || TOP10 || Cm 12,5<br />
|-<br />
|<partinfo>BBa_J23106</partinfo>_4C5 || TOP10 || Cm 12,5<br />
|-<br />
|<partinfo>BBa_J23110</partinfo>_4C5 || TOP10 || Cm 12,5<br />
|-<br />
|<partinfo>BBa_J23114</partinfo>_4C5 || TOP10 || Cm 12,5<br />
|-<br />
|<partinfo>BBa_J23116</partinfo>_4C5 || TOP10 || Cm 12,5<br />
|-<br />
|<partinfo>BBa_J23118</partinfo>_4C5 || TOP10 || Cm 12,5<br />
|}<br />
<br />
Plates were incubated at 37°C overnight.<br />
<br />
Inoculum of self inducible promoters for TECAN test:<br />
<br />
{| border='1'<br />
| I7 ||I8 || I9 || I10<br />
|-<br />
| I12 || I8_4C5 || I15 || I16<br />
|-<br />
| I17 || I18|| I19 || I27<br />
|-<br />
| I28 || I30|| <partinfo>BBa_B0032</partinfo> || A2 (<partinfo>BBa_J23101</partinfo>+GFP)<br />
|}<br />
<br />
----<br />
After 30 hours, Sudan Black staining protocol was performed on 70ul cultures and 5 microscope slides were prepared. The resulting images are shown here:<br />
<br />
{|align="center"<br />
|-<br />
|[[Image:UNIPV10_PBHR68_nothing_30.jpg|thumb|200px|center|PBHR68 with nothing added in the culture, after 30 hours]] || |[[Image:UNIPV10_PBHR68_nothing_2_30.jpg|thumb|200px|center|PBHR68 with nothing added in the culture, after 30 hours]]<br />
|-<br />
|[[Image:UNIPV10_RBS32_nothing_30.jpg|thumb|200px|center|<partinfo>BBa_B0032</partinfo> with nothing added in the culture, after 30 hours (negative control)]] || |[[Image:UNIPV10_RBS32_nothing_2_30.jpg|thumb|200px|center|<partinfo>BBa_B0032</partinfo> with nothing added in the culture, after 30 hours (negative control)]] <br />
|-<br />
|[[Image:UNIPV10_PBHR68_gly_30.jpg|thumb|200px|center|PBHR68 with 2% glycerol added in the culture, after 30 hours]] || |[[Image:UNIPV10_PBHR68_gly_2_30.jpg|thumb|200px|center|PBHR68 with 2% glycerol added in the culture, after 30 hours]]<br />
|-<br />
|[[Image:UNIPV10_PBHR68_IPTG_30.jpg|thumb|200px|center|PBHR68 with 1mM IPTG added in the culture, after 30 hours]] || |[[Image:UNIPV10_PBHR68_IPTG_2_30.jpg|thumb|200px|center|PBHR68 with 1mM IPTG in the culture, after 30 hours]]<br />
|-<br />
|[[Image:UNIPV10_PBHR68_gly_IPTG_30.jpg|thumb|200px|center|PBHR68 with 1mM IPTG and 2% glycerol added in the culture, after 30 hours]] || |[[Image:UNIPV10_PBHR68_gly_IPTG_2_30.jpg|thumb|200px|center|PBHR68 with 1mM IPTG and 2% glycerol added in the culture, after 30 hours]]<br />
|}<br />
<br />
----<br />
Inoculum of<br />
*<partinfo>BBa_J13002</partinfo><br />
*I33<br />
*I34<br />
*I39<br />
*I41<br />
in 5 ml LB+Amp<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
<br />
==August, 27th==<br />
Previously inoculated strains were miniprepped and quantified as follows:<br />
*<partinfo>BBa_J13002</partinfo>: 29,9 ng/ul<br />
*I33: 171,6 ng/ul<br />
*I34: 219,4 ng/ul<br />
*I39: 21,2 ng/ul<br />
*I41: 94,7 ng/ul<br />
<br />
Digestions (3,5 hours) for ligations:<br />
*I47: <partinfo>BBa_J13002</partinfo> (S-P) + I33 (X-P)<br />
*I48: I39 (S-P) + I34 (X-P)<br />
*I49: I39 (S-P) + I41 (X-P)<br />
<br />
{| border="1" align='center'<br />
| ''Culture'' || ''Kind'' || ''Final reaction volume (ul) '' || ''DNA (ul)'' || ''H20 (ul)'' || ''Enzyme 1 (ul)'' || ''Enzyme 2 (ul)'' || ''Buffer H (ul)'' <br />
|-<br />
| <partinfo>BBa_J13002</partinfo> || Vector || 25 || 21,5 || 0 || 0,5 SpeI || 0,5 PstI || 2,5<br />
|-<br />
| I39 || Vector || 25 || 21,5 || 0 || 0,5 S || 0,5 P || 2,5<br />
|-<br />
| I33 || Insert || 25 || 10,5 || 11 || 0,5 XbaI || 0,5 P || 2,5<br />
|-<br />
| I34 || Insert|| 25 || 13,5 || 8 || 0,5 X || 0,5 P || 2,5<br />
|-<br />
| I41 || Insert || 25 || 2 || 19,5 || 0,5 X || 0,5 P || 2,5<br />
|}<br />
<br />
Samples were loaded into a medium gel, run and cut<br />
[[Image:UNIPV10_27_8_10_digest_step2A.jpg|thumb|300px|center|Samples gel run/cut]]<br />
<br />
Gel extraction was quantified as follows:<br />
*<partinfo>BBa_J13002</partinfo> (S-P): 19,9 ng/ul<br />
*I33 (X-P): 17,7 ng/ul<br />
*I34 (X-P): 11,1 ng/ul<br />
*I39 (S-P): 4,7 ng/ul<br />
*I41 (X-P): 6,2 ng/ul<br />
<br />
Ligation of<br />
*I47: <partinfo>BBa_J13002</partinfo> (S-P) + I33 (X-P)<br />
*I48: I39 (S-P) + I34 (X-P)<br />
*I49: I39 (S-P) + I41 (X-P)<br />
<br />
<br />
----<br />
<br />
Plates containing the transformed ligations were all grown. Colony PCR was performed to screen if the length of insert was correct.<br />
<br />
{| align='center' border='1'<br />
| '''ligation name'''|| '''number of colonies screened'''<br />
|-<br />
| <partinfo>BBa_J23100</partinfo>_4C5 || 2 colonies<br />
|-<br />
| <partinfo>BBa_J23101</partinfo>_4C5 || 2 colonies<br />
|-<br />
| <partinfo>BBa_J23105</partinfo>_4C5 || 2 colonies<br />
|-<br />
| <partinfo>BBa_J23106</partinfo>_4C5 || 2 colonies<br />
|-<br />
| <partinfo>BBa_J23110</partinfo>_4C5 || 2 colonies<br />
|-<br />
| <partinfo>BBa_J23114</partinfo>_4C5 || 1 colony<br />
|-<br />
| <partinfo>BBa_J23116</partinfo>_4C5 || 2 colonies<br />
|-<br />
| <partinfo>BBa_J23118</partinfo>_4C5 || 2 colonies<br />
|}<br />
<br />
<br />
[[Image:UNIPV10_screening_promoters_Anserson_low_copy.jpg|thumb|300px|center|PCR screening for Anderson Promoters transferred in low copy plasmid <partinfo>pSB4C5</partinfo>]]<br />
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Gel results show that we have the correct clones for <partinfo>BBa_J23100</partinfo>_4C5, <partinfo>BBa_J23101</partinfo>_4C5, <partinfo>BBa_J23105</partinfo>_4C5, <partinfo>BBa_J23106</partinfo>_4C5, <partinfo>BBa_J23110</partinfo>_4C5, <partinfo>BBa_J23114</partinfo>_4C5. Glycerol stocks were prepared for these parts and are stored at -80°C. <br />
<br />
<partinfo>BBa_J23116</partinfo>_4C5 and <partinfo>BBa_J23118</partinfo>_4C5 need to be further screened, so we will pick other colonies next week and we will perform a new colony PCR.<br />
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<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test27agosto|Tecan Test]]</font> was performed on prepared samples, after the usual protocol (dilution, medium change and dilution 1:1000).<br />
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Transformation of I29 in ''E. coli'' TOP10<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
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==August, 28th==<br />
Ligations I47, I48, I49 were stored at +4°C.<br />
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I29 plate was stored at +4°C.<br />
<div align="right"><small>[[#indice|^top]]</small></div><br />
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