Part:BBa_K2476002
Mutant CheY + Dronpa fusion protein regulated by IPTG
Uses an IPTG inducible promoter with RBS (BBa_J04500), Mutant CheY + Linker/Dronpa/Linker at 60-61 (BBa_K2476014), Dronpa (BBa_K2476010), and a double terminator (BBa_B0015). Will express Mutant CheY + Dronpa fusion protein when induced with IPTG. This part contains the sequence for a constitutively active CheY protein. When the chemotaxis protein CheY is phosphorylated, it becomes capable of binding to the flagellar motor protein FliM. This then results in clockwise rotation of the flagella on the bacterial cell and tumbling behavior occurs. In this part, we've induced a mutation in the CheY protein so that it behaves as though it is constitutively phosphorylated so that it will continuously bind to the FliM protein to produce a tumbling phenotype within the bacterial cell. In addition to the constitutively active CheY protein, we've also added two dronpa monomeric units that flank the active site on the CheY protein where it binds to FliM to produce the tumbling phenotype. The dronpa proteins are photoswitchable, meaning that they will dimerize under 500nm light and block the active site of CheY, effectively preventing it from binding to FliM. This should result in a running phenotype within the bacterial cell. On the other hand, under exposure to 400nm light, the dronpa dimers will once again monomerize and cause the exposure of the CheY protein's active site and once again induce a tumbling phenotype because the mutant form of CheY acts as if it was constitutively phosphorylated. This part can be used to control chemotaxis of bacteria via LED light sources of the wavelengths stated above.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 419
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 368
- 1000COMPATIBLE WITH RFC[1000]
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