The idea behind the project is fluorescence complementation, which involves the joining of two leucine zipper proteins Jun and Fos each fused to a half terminus of YFP.
Subsequently, both of these chimeric proteins were fused to a beta gene that codes for a membrane protein. The project involved performing a PCR reaction to produce two inserts, the
N-terminus and the C-terminus of the YFP, and then ligating these inserts into 2 vectors, containing the
Jun-beta and the Fos-beta respectively.
We transformed two cell populations, one expressing Jun-beta-YFPN and the other expressing Fos-beta-YFPC.These two cell populations were combined and the two vectors were then expressed, ideally resulting in the fusion of the Jun and Fos leucine zipper proteins on the cell membrane when the cells are in close contact. This would result in the binding of the two halves of the YFP protein resulting in flourescence.
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The Elowitz Repressilator attempts to decrease the loss of standard oscillations that previous repressilators faced by utilizing quorum sensing as a means of synchronizing and maintaining standard oscillations. We expanded on this theory by adding YFP and CFP to allow a visual confirmation of the oscillation, and a TetR promoter infront of the LuxR gene and cI after the LuxI gene. Our hopes were that this would assist in standardizing the oscillation of the bacteria.
Oscillator Example
Theory Behind the Oscillator
Methods and Materials
Results
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