Team:EPF-Lausanne/Our Project/Summary

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The mutants were created by site-specific and PCR-induced mutagenesis; we introduced point mutations at key amino acids involved in DNA recognition, in an attempt to alter the specificity of the TetR mutants.
The mutants were created by site-specific and PCR-induced mutagenesis; we introduced point mutations at key amino acids involved in DNA recognition, in an attempt to alter the specificity of the TetR mutants.
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''' 2) determining the binding energy landscape of each mutants
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This is the ''in vitro'' characterization, which was performed thank to a microfluidic chip. This technique, called MITOMI, allows parallel testing of one mutant with 700(?) different DNA sequences. The absolute binding energy is then measured, and a weblogo can be generated.
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''' 3) determining the binding of each mutant to the wild-type TetO sequence ''in vivo''
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For this characterization step, we set up a reporter construct: TetR constitutively expressed and RFP with a wild-type Ptet promoter. The more RFP is expressed in the cells, the less the TetR mutant recognizes Ptet.
{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Revision as of 07:28, 28 October 2011