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Team:INSA Toulouse/contenu/lab practice/results/blue sensor
From 2013.igem.org
Results - Blue Light Sensor Characterization
Objective
Characterize the blue light sensor system using YF1-FixJ with its promoter FixK2. Reminder: in darkness, YF1-FixJ can bind to the FixK2 promoter and actives the downstream coding sequence whereas with blue light, YF1-FixJ cannot bind to the FixK2 promoter, impeaching transcription.Conception
The following construction was designed:
YF1 and FixJ genes were under the control of the pTet promoter, the general inducer system to mimic the real behavior of the blue 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 blue light conditions, expression was supposedly promoted, except when aTc was added in the media.
Result
We did not obtain the previous construction but managed to construct a biobrick to characterize the blue light sensor system. YF1 and Fix J were placed under the control of a weak promoter.
Used parts are available here :
- Promoter J23116 (http://parts.igem.org/Part:BBa_J23116)
- YF1-FixJ (http://parts.igem.org/Part:BBa_K592016)
- Terminator B1010 (http://parts.igem.org/Part:BBa_B1010)
- FixK2 promoter (http://parts.igem.org/Part:BBa_K592006)
- RFP (http://parts.igem.org/Part:BBa_K081014)
The transformed strain was analyzed as follows:
Several subcultures were done to ensure that transformed bacteria were placed in light/dark states for at least 3 subcultures (1000) generation. Visually, cultures in darkness didn’t show any differences with these placed in the light. Fluorescence measurements during induction for 18 hours demonstrated the same inconclusive results.
Discussion
Our interpretation of the non-expression of RFP is that the expression of YF1 and FixJ was too low to activate the FixK2 promoter. A biobrick without promoter in front of YF1 and FixJ was also submitted to the registry: YF1-FixJ-terminator-pFixK2-RFP (BBa_K1132020). Adding a strong promoter may improve the system.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
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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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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