Team:Paris Bettencourt/Improve



Dronpa is a reversible photoswitchable fluorescent protein that is switched on by default “fluorescent” and is switched off when illuminated by cyan light (~500nm). Dronpa Fluorescence is recovered by shining violet light (~400nm). And has been used in a design that facilitates the optical control of protein activities This part contains a device of two copies of Dronpa Fluorescent Protein that are codon optimized for E Coli with two BsaI cutting site in between to allow the insertion of various proteins to be tested. This coding sequence of the 2 dronpa domains has 2 mutations I4V and R149H in the first dronpa domain and an F78S mutation in the second domain that were obtained by error prone PCR. This mutant version of Dronpa has shown a better performance than the wild type in controlling the activity of both TetR [fig2] and β-galactosidase[fig 4], The conditions for testing the repressors is indicated in figure 1 while the workflow of the β-galactosidase activity experiment is indicated in figure 3

Figure 1 : the experiment conducted with the repressors caged with Dronpa Figure 2 :Results of testing the activity of TetR caged with Dronpa with our library of synthetic operators. Testing with TetR caged with either wt-Dronpa (BBa_K2510108) or a mutated version(BBa_K2510109) Figure 3: An overview of the experiment done to evaluate the activity of β-galactosidase-Dronpa fusion. Figure 4: Top: X-Gal grayscale picture, testing the activity of β-galactosidase fusion with both wtDronpa and mutDronpa, indicating that β-galactosidase-mutDronpa fusion is more responsive to cyan light than the β-galactosidase-wtDronp. Down:90 fold difference in the activity between the MutDronpa caged beta-gal open and closed state after 4 hours of incubation

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