Team:EPF-Lausanne

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== Project summary ==
== Project summary ==
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Our goal is to design transcription factors (TFs) that bind to new sequences, currently unavailable to synthetic biologists. These would allow to build larger genetic circuits than currently possible, with more independently regulated genes. The new transcription factors will be derived from tetR, one of the better characterised TFs out there. To characterise the new ones, we will introduce the tetR mutants and their corresponding mutant promoters into the circuit illustrated below, containing a LacI inverter and a reporter gene. The reporter for ''in vitro'' experiments is a fluorescence gene such as RFP, and a lysis system for ''in vivo'' experiments. The latter lyses cells with effective transcription factors, so that exclusively their DNA can be recovered.
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Our project this summer was to set up a high-throughput method to create and characterize new transcription factors (TFs) that would recognize different promoter sequences. We mutated the TetR transcription factor and characterized <i>in vitro</i> their affinity to the consensus sequence (the Ptet promoter) as well as their position-weight matrices. The next step of the characterization is an <i>in vivo</i> readout system; we created and tested 2 different readout systems based on RFP expression, with either a positive or negative selection of the TetR-Ptet interaction. Wanting a high-throughput method, we decided also to use a lysis cassette as a reporter gene. The idea is to kill the cells in which a TetR mutant recognizes a Ptet sequence (either the consensus or a mutated one) and to recover their DNA; this can be coupled to microfluidics chemostat chambers, where hundreds of cell colonies can be grown at the same time.
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Our goal is to design transcription factors (TFs) that bind to new sequences, currently unavailable to synthetic biologists. These would allow to build larger genetic circuits than currently possible, with more independently regulated genes. The new transcription factors will be derived from tetR, one of the better characterised TFs out there. To characterise the new ones, we will introduce the tetR mutants and their corresponding mutant promoters into the circuit illustrated below, containing a LacI inverter and a reporter gene. The reporter for ''in vitro'' experiments is a fluorescence gene such as RFP, and a lysis system for ''in vivo'' experiments. The latter lyses cells with effective transcription factors, so that exclusively their DNA can be recovered. -->
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[[File:EPFL_Summary_(with_TFs).png|700px]]
 
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Revision as of 21:07, 18 September 2011