Part:BBa_K2510102
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
- 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
| None |