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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.