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Team:INSA Toulouse/contenu/lab practice/results/ribo
From 2013.igem.org
Results
Riboswitches Characterization
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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