Difference between revisions of "Part:BBa K2510102"
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<partinfo>BBa_K2510102 short</partinfo> | <partinfo>BBa_K2510102 short</partinfo> | ||
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| − | <p>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 | + | <h1> BBa_K1680006 </h1> |
| + | <div class=text1><p> | ||
| + | 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 </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. |
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</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 4: </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> | ||
</html> | </html> | ||
Latest revision as of 03:38, 16 December 2017
pdDronpa mutant
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 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
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 755
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 758
Illegal BsaI.rc site found at 752