Latest revision as of 11:42, 5 August 2011

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Plasmids strategy

We are building a system containing TetR and LacI, that control the expression of a selection gene. The sequential use of these two repressors allows us to have a direct activation of the selection gene when TetR binds its recognition sequence.

The selection gene is either RFP or a lysis cassette. RFP allows direct recognition of correctly assembled plasmids, which is also useful to see if the transformation has been efficient. Furthermore, it is an easy way to detect TetR binding. The lysis cassette will be useful for chemostat cultures, allowing us to recover DNA from mutants in which TetR binds to a sequence (either consensus sequence or modified one). We hope that the chemostat chambers will allow high-throughput in vivo selection of our mutants.

We need several plasmids to build our entire system. We chose a 2-plasmid strategy that looks like this:

The TetR plasmid containing TetR under a constitutive promoter
The reporter plasmid containing a reporter gene (either RFP or a lysis device) and a LacI inverter for tetR.

@@ Line 1: / Line 1: @@
 {{:Team:EPF-Lausanne/Templates/Header|title=Plasmids strategy}}
+We are building a system containing TetR and LacI, that control the expression of a selection gene. The sequential use of these two repressors allows us to have a direct '''activation''' of the selection gene when TetR '''binds''' its recognition sequence.
-We are using TetR and LacI to inverse the repression, which allows positive selection. We will select at two levels: in vivo and in vitro, using either lysis cassette or RFP respectively:
+[[File:EPFL_Summary_(with_TFs).png|650 px]]
-* in vitro: We can test the binding of TetR directly on plates by measuring RFP fluorescence
-* in vivo: The cells will grow on chemostat chips. If TetR binds to the recogintion sequence, the cells will lyse and we will be able to recover the DNA
-We have one plasmid with the TetR gene, where we will introduce mutations and two plasmids for in vivo or in vitro selection:
+The selection gene is either RFP or a lysis cassette. RFP allows direct recognition of correctly assembled plasmids, which is also useful to see if the transformation has been efficient. Furthermore, it is an easy way to detect TetR binding. The lysis cassette will be useful for chemostat cultures, allowing us to recover DNA from mutants in which TetR binds to a sequence (either consensus sequence or modified one). We hope that the chemostat chambers will allow high-throughput <i>in vivo</i> selection of our mutants.
+We need several plasmids to build our entire system. We chose a 2-plasmid strategy that looks like this:
+* The '''TetR plasmid''' containing TetR under a constitutive promoter
+* The '''reporter plasmid''' containing a reporter gene (either RFP or a lysis device) and a LacI inverter for tetR.
-[[File:EPFL_Plasmids.png|300 px]]
+[[File:EPFL_Plasmids.png|700 px]]
-[[File:EPFL_Plasmids(RFP_instead_of_Lysis).png|300 px]]
+[[File:EPFL_Plasmids(RFP_instead_of_Lysis).png|700 px]]
 {{:Team:EPF-Lausanne/Templates/Footer}}

Team:EPF-Lausanne/Our Project/Plasmids strategy

From 2011.igem.org

Latest revision as of 11:42, 5 August 2011

Plasmids strategy