Part:BBa_K4138005:Design
VVD-CMI
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 226
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 226
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 226
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 226
Illegal AgeI site found at 90 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
CMI and VVD are both homodimers, where VVD has been fused to the N-terminus of the CMI chain. Thus, both CMI chains have a fused VVD. The goal was for VVD to be a monomer in the dark, preventing dimerization and thus phosphate binding of CMI. Conversely, VVD would be a dimer in the light, and this would promote dimerization of CMI and allow phosphate binding. We hypothesize that the formation of a disulfide bridge would stabilize the CMI dimer so that the fusion protein itself would be a dimer in the absence of phosphate. To prevent this, the disulfide bridge that would be formed intermolecularly between the dimers, that is, between C30 of one chain and C106 of the other chain, was disrupted by exchanging cysteines with serine. We tended to C30 because it is located at the beginning of the first helix and only one loop comes before. C106 is located between two structure-giving elements, so an exchange would be more invasive.
Source
The individual sequences of the construct components can be found in the following parts:
VVD: BBa_K4138002
CMI: BBa_K4138003
Linker: BBa_K4138006