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Team:EPF-Lausanne/Strategy
From 2009.igem.org
Contents |
Cloning strategy
Our aim was to create a biobrick containing LovTAP under the influcence of an inducible promoter. This part needed to contain (in order) the inducible promoter (LacI), RBS (ribosome binging site), the LovTAP gene (that we recovered from Dr. ?? Sosnick’s lab) and finally a terminator (Term). This entire part was of course to be flanked by the standard prefix (E,X) and suffix (S,P). In order to synthesize this part, we proceeded in three steps :
1. We ligated LovTAP with the terminator (we used a double terminator).
2. We also ligated LacI with RBS.
3. Finally we ligated these two parts together to obtain our biobrick.
So finally we have :
With the biobrick, we have an inducible system : when we add IPTG, the promoter activates the expression of the LovTAP gene.
Read out system
Once we have our protein LovTAP produced, we needed a read out system to assess whether the protein was functional. We designed two different read out systems :
1. The read out 1 (RO1) contains the tryptophan operon followed by RBS, RFP and Term. In normal conditions, RFP should be expressed (so we should see some red fluorescence). This is because TRP repressor (TrpR) is expressed only if there is tryptophane available in the medium and then it inhibits TRP operon. The idea is that when LovTAP is produced (and in light state, that is when we add blue light), it will bind to the TRP operon and repress the RFP gene. We should therefore observe a decrease in red fluorescence. In order to characterize the read out, we tested it in different conditions : with or without TRP, with different amount of TRP, with or without light. If add TRP, we expect that the fluorescence will decrease.
2. The read out 2 (RO2) is composed of TRP op, RBS, TetR, Term and then TetRP, RBS, RFP and Term. It is a double repressor system : TrpR/LovTAP binds to the TRP operon, so repressing the expression of TetR. On the other hand, TetR (if expressed) inhibits the prodcution of RFP by acting on the TetR promoter. The final result is that if LovTAP is active, red fluorescence should increase as RFP is expressed. TRP has the same effect as LovTAP but we also used ATC (anhydrotetracyclin) : ATC has the same effect as TRP, when it binds to TetR it prevents binding to its promoter sequence TetO ! When we add TRP and ATC, we should therefore have the same effect as with an active LovTAP : production of red fluorescence.
So all this function like this :
