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Team:INSA Toulouse/contenu/lab practice/results
From 2013.igem.org
Results
Characterizations
Carry
General Inducer
Blue Light Sensor
Red Light Sensor
Riboswitches
Logic Gates
Recombinases
T7 Polymerase
XOR1
In vitro
Description des conditions d'expérimentation
In vitro recombinase characterization protocol
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In vivo
Description des conditions d'expérimentation
In vitro recombinase characterization protocol
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AND1
In vitro
Description des conditions d'expérimentation
In vitro recombinase characterization protocol
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In vivo
Description des conditions d'expérimentation
In vitro recombinase characterization protocol
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Red Light Sensor
In vitro
Objective
Characterize the red light sensor system using Cph8 with its promoter pOmpc. Reminder: in darkness, the EnvZ histidine kinase of Cph8 phosphorylates endogenous OmpR, a transcription factor which activates transcription from the OmpC promoter. Under red light conditions, the activity of the EnvZ histidine kinase domain is inhibited, impeaching transcription from the downstream sequence.
Conception
The following construction was designed:
Cph8 gene was under the control of the pTet promoter, the general inducer system to mimic the real behavior of the red light sensor system in the E. calculus design (https://2013.igem.org/Team:INSA_Toulouse). The transformed strain was supposed to express the modified RFP while in the dark. In red light conditions, expression was supposedly promoted, except when aTc was added in the media.
Result
We obtain the previous construction to characterize the red light sensor system.
Used parts are available here :
- Strong promoter and strong rbs (http://parts.igem.org/Part:BBa_K608002)
- Ho1 (http://parts.igem.org/Part:BBa_I15008)
- PcyA (http://parts.igem.org/Part:BBa_K081017)
- TetR (http://parts.igem.org/Part:BBa_P0440)
- pOmpC promoter (http://parts.igem.org/Part:BBa_R0082)
- rfp (http://parts.igem.org/Part:BBa_K081014)
- ptet (http://parts.igem.org/Part:BBa_R0040)
- Cph8 (http://parts.igem.org/Part:BBa_K592018)
Discussion
We succeeded in cloning the construction (http://parts.igem.org/wiki/index.php?title=Part:BBa_K1132017), but did not characterize it due to lack of time. The final biobrick must be used in an E. coli deficient in wild-type EnvZ. The delta EnvZ strain was obtained from the Paris Bettencourt team and has been already described by many other iGEM projects.
http://www.nature.com/nature/journal/v438/n7067/abs/nature04405.html
Description des conditions d'expérimentation
In vitro recombinase characterization protocol
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Blue Light Sensor
In vitro
Description des conditions d'expérimentation
In vitro recombinase characterization protocol
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In vivo
Description des conditions d'expérimentation
In vitro recombinase characterization protocol
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Pol T7
In vitro
Description des conditions d'expérimentation
In vitro recombinase characterization protocol
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In vivo
Description des conditions d'expérimentation
In vitro recombinase characterization protocol
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General inductor
In vivo
The couple TetR-pTet system construction is a first step to characterize the entire system. A constitutive promoter (BBa_J23116) was assemble to tetR and then assemble to pTet-rbs-RFP-term (Bba_I13521). pTet is a regulator constitutively ON that expresses the mRFP1 protein. TetR is an inducer that binds to pTet promoter and thus, represses expression the downstream system. Supply of tetracycline or its analog aTc (anhydrotetracycline) is known to bind to tetR and invert the operation (inhibits expression of mRFP in this case).
After 18 hours, clones containing this assembly (BBa_J23116-TetR-pTet-RFP) show a leaky basal expression of mRFP. We suppose that the promoter was too weak to express TetR in large quantity. Assembly of a stronger promoter could improve the system and lock the response to an ON/OFF response.
Besides, an experience was done to analyze the effect of the aTc inducer. Result show a visible induction of the red fluorescent protein expression by addition of aTC (60 ng/mL).
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