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Team:EPF-Lausanne/Our Project/Reporter Systems
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Our overall strategy to develop new transcription factors includes an ''in vivo'' characterization of the affinity and specificity of our TetR mutants. To that end, we set up two reporter systems based on RFP fluorescence. | Our overall strategy to develop new transcription factors includes an ''in vivo'' characterization of the affinity and specificity of our TetR mutants. To that end, we set up two reporter systems based on RFP fluorescence. | ||
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| + | The first reporter system consists of a TetR-RFP cascade. It has been successfully used to characterize some TetR mutants. Our second reporter system is a longer cascade, made up of TetR, LacI and RFP. Whereas in the first system RFP is expressed in the '''absence''' of a TetR-Ptet interaction, in the second one, the TetR-Ptet interaction '''directly induces''' RFP expression. | ||
| + | The affinity of each mutant can be deduced from the RFP expression profile induced in our reporter system. The specificity can be assessed by coupling each mutant to different Ptet sequences. | ||
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| + | To set up and characterize our two reporter systems, we assembled several plasmids that are to be co-transformed in order to create the whole system. We also had to characterize the two different promoters we used to control RFP expression: an existing Ptet promoter and a new Plac promoter. It is important to know the intrinsic strength of these promoters in order to be able to properly analyze the data generated by our two reporter systems. | ||
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| + | [[File:InVivo.png|700px]] | ||
{{:Team:EPF-Lausanne/Templates/Footer}} | {{:Team:EPF-Lausanne/Templates/Footer}} | ||
Latest revision as of 19:10, 25 October 2011
In Vivo Characterization
In vivo Main | TetR-RFP System | TetR-LacI-RFP System | Ptet Characterization | Plac Characterization | Plasmid DetailsOur overall strategy to develop new transcription factors includes an in vivo characterization of the affinity and specificity of our TetR mutants. To that end, we set up two reporter systems based on RFP fluorescence.
The first reporter system consists of a TetR-RFP cascade. It has been successfully used to characterize some TetR mutants. Our second reporter system is a longer cascade, made up of TetR, LacI and RFP. Whereas in the first system RFP is expressed in the absence of a TetR-Ptet interaction, in the second one, the TetR-Ptet interaction directly induces RFP expression. The affinity of each mutant can be deduced from the RFP expression profile induced in our reporter system. The specificity can be assessed by coupling each mutant to different Ptet sequences.
To set up and characterize our two reporter systems, we assembled several plasmids that are to be co-transformed in order to create the whole system. We also had to characterize the two different promoters we used to control RFP expression: an existing Ptet promoter and a new Plac promoter. It is important to know the intrinsic strength of these promoters in order to be able to properly analyze the data generated by our two reporter systems.
