Part:BBa_K4390003
merR mutated expression construct
BBa_K4390003 is a working transcriptional unit which expresses a mutant MerR repressor (BBa_K1724002). It is composed of the constitutive promoter J23100 (BBa_J23100), a ribosome binding site (BBa_B0034), the mutant MerR repressor sequence (BBa_K1724002) and the weak synthetic L2U2H09 Terminator (BBa_K4390001). This unit was assembled using JUMP and can be assembled into a pJUMP29-1A KanR Type IIS Level 1 vector plasmid for expression in cells.
Usage and Biology
We used the mutant MerR repressor as the wild-type MerR regulatory protein that will normally regulate mercury resistance operons in Gram-negative bacteria, however, this protein was subjected to directed evolution and produced a mutant which responds to cadmium ions rather than mercury. This mutant MerR will bind to a Mer promotor in the absence of cadmium ions. We used this transcriptional unit alongside the Cd biosensor (BBa_K4390050) so that when we use cell lysate which expresses our transcriptional unit there would be mutant MerR present in the lysate. When this cell-free extract is combined with the Cd biosensor it induces transcriptional repression of the linear biosensor when cadmium ions are not present. When cadmium ions are present the mutant MerR binds to the metal ions and allows transcription of the biosensor and would therefore generate a fluorescent output. It should be noted that T7 RNA polymerase, chemical energy (ATP), NTPs and the DFHBI are also required in the cell-free reaction so that fluorescence is observed. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 11
Illegal NheI site found at 34 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 317
Illegal AgeI site found at 419 - 1000COMPATIBLE WITH RFC[1000]
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