Team:EPF-Lausanne/Our Project/Plasmids strategy

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{{:Team:EPF-Lausanne/Templates/Header|title=Plasmids strategy}}
{{:Team:EPF-Lausanne/Templates/Header|title=Plasmids strategy}}
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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.
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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.
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[[File:EPFL_Summary_(with_TFs).png|650 px]]
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[[File:EPFL_Summary_(with_TFs).png|700 px]]
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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:
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* in vitro: We can test the binding of TetR directly on plates by measuring RFP fluorescence
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* 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
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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:
We need several plasmids to build our entire system. We chose a 2-plasmid strategy that looks like this:
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* One plasmid containing TetR under a constitutive promoter
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* The '''TetR plasmid''' containing TetR under a constitutive promoter
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* One plasmid containig LacI under TetR repression and RFP or lysis under LacI repression
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* The '''reporter plasmid''' containing a reporter gene (either RFP or a lysis device) and a LacI inverter for tetR.
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[[File:EPFL_Plasmids.png|700 px]]
[[File:EPFL_Plasmids.png|700 px]]

Latest revision as of 11:42, 5 August 2011

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