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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. Having both constructs will allow us to select at two levels: in vivo and in vitro:

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

• One plasmid containing TetR under a constitutive promoter
• One plasmid containig LacI under TetR repression and RFP or lysis under LacI repression