Difference between revisions of "Team:Paris Bettencourt/Improve"
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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 | 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 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 | + | 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 </p> |
| + | <img src="https://static.igem.org/mediawiki/2017/b/b1/96_conditions.png"> | ||
| + | <b>Figure 1 : </b> the experiment conducted with the repressors caged with Dronpa | ||
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| + | </span> | ||
| − | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/2/22/TetR_dronpa.png"> |
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<span class="image-span text-center"> | <span class="image-span text-center"> | ||
| − | <b>Figure | + | <b>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) </span> |
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<span class="image-span text-center"> | <span class="image-span text-center"> | ||
| − | <b>Figure | + | <b>Figure 3: </b> An overview of the experiment done to evaluate the activity of β-galactosidase-Dronpa fusion. |
| − | + | ||
</span> | </span> | ||
| − | <img id=" | + | <img id="fig14" src="https://static.igem.org/mediawiki/2017/0/01/Aya_figure_14.png" /> |
| − | + | <img src="https://static.igem.org/mediawiki/2017/3/3f/Lacz_mutDronpa.png"> | |
<span class="image-span text-center"> | <span class="image-span text-center"> | ||
| − | <b>Figure | + | <b>Figure 14: </b> 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 | |
</span> | </span> | ||
</div> | </div> | ||
Revision as of 02:53, 16 December 2017
IMPROVED PARTS
BBa_K1680006
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 14: 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
Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
bettencourt.igem2017@gmail.com
bettencourt.igem2017@gmail.com 
