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Team:UNIPV-Pavia/Project/PromotoriAuto
From 2010.igem.org
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| - | '''Discussion''': two | + | '''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: |
*varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation) | *varying the strength of the promoter controlling the production of the signal molecule (Sender Modulation) | ||
*varying the copy number of vectors bearing both Sender and Receiver circuits | *varying the copy number of vectors bearing both Sender and Receiver circuits | ||
*varying the growth medium (LB or M9) | *varying the growth medium (LB or M9) | ||
| - | + | A graphical summary is reported in the figures below: | |
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|[[Image:pv_SwitchPointLB.png|700px|thumb|center| Typical ''E. coli'' growth curve with O.D.start evaluated by Threshold algorithm in LB]] | |[[Image:pv_SwitchPointLB.png|700px|thumb|center| Typical ''E. coli'' growth curve with O.D.start evaluated by Threshold algorithm in LB]] | ||
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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. | 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. | ||
| - | In | + | 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). |
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| - | 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 | + | 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. |
| - | For RPU values greater or equal to 1, the expected behaviour is not confirmed anymore. This is probably | + | 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. |
| - | + | ||
| - | The combination of promoter strength variation and plasmid copy number modulation allows the creation of a library of self-inducible devices | + | 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. |
<div align="right"><small>[[#indice|^top]]</small></div> | <div align="right"><small>[[#indice|^top]]</small></div> | ||
Latest revision as of 21:41, 27 October 2010
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Self-inducible promotersRegulation of signal protein productionExperimental 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:
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 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:
It was not possible to evaluate promoters activities in low copy number plasmids in LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2. Data were analyzed as reported in Data analysis for RPU evaluation; Results: results are shown here.
Doubling times were evaluated for the described cultures (HC stands for High Copy and LC stands for Low Copy):
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. 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:
Some of the promoters could not be cloned upstream of these devices because they produced amounts of LuxI protein that give a high metabolic burden for E. coli, so it was not possible to study all the combinations as transformants could not be obtained in some cases. 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. Quantification of the HSL produced by autoinducer generatorsExperimental 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 this section. <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. 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:
The amount of 3OC6-HSL produced by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> after a 6 hour cell growth is reported in Fig.9 and in the table below:
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. Modulation of plasmid copy numberSignal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) in high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high copy number plasmid (<partinfo>pSB1A2</partinfo>). The circuits we obtained and tested are summarized in tables.
Sender/Receiver devices assembled as a unique BioBrick part on the same vector
Sender devices assembled on low copy number vector and Receiver device on high copy number vector
ResultsThe following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:
Cultures of E. coli TOP10 bearing the plasmids containing the self-inducible devices expressing GFP were grown according to this protocol and all data collected were analyzed as explained in 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.
For every self-inducible device, several parameters were evaluated:
Results are summarized in the following tables:
Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>
Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>
LEGEND OF TABLES: 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. *: 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. **: 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. <partinfo>BBa_K300016</partinfo> is labelled with *, 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). 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:
A graphical summary is reported in the figures below:
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. 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).
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. 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. 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.
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