Part:BBa_K1075027
SplitSspB(Rapamycin inducable)-pLac-RBS34-mCherry-(Ec)ssrA(DAS+4)-TT
construction
The part contains the promoter plac, a ribosomal binding site, the red fluorescent protein mCherry fused to the E. coli(ec)ssrA (DAS+4) tag and a double terminator. It is integrated in the plasmid pJD427, which contains a ecSspB split system.
biology
The plasmid pJD427 contains the two parts of splitted ecSspB, which can be fused (and thus regain function) by addition of rapamycin. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220803/]
plac is a IPTG inducible promoter.
The ribosomal binding side is a sequence of mRNA where the ribosome binds to start translation.
The ecssrA tag is a short peptide sequence, which is fused to the C-terminus of proteins, which should be degraded. It relates to the protease ClpXP complex in E.coli and it also allows the adaptor protein ecSspB binding and delivering substrates to the proteases in order to make the process more efficient. The mutated ecssrA(DAS+4) weakens a direct binding between proteases and ecssrA(DAS+4) and increases the dependance of ecsspB. [http://www.ncbi.nlm.nih.gov/pubmed/16762842]
mCherry is a red fluorescent protein with the excitation maximum at 587 nm and the emission maximum at 610 nm. [http://www.ncbi.nlm.nih.gov/pubmed/15558047]
The double terminator stops the transcription at this point.
application
As we want to control protein degradation by controlling the function of ecSspB, we tag the red fluorescent protein mCherry with ecssrA(DAS+4) to measure the degradation rate. We control ecSspB by splitting it into two parts each of which cannot induce degradation on its own but regains function by addition of rapamycin. As the co-transformation of the two plasmids didn’t work, we combined them to one.
Application as a bacterial fotographic film might be possible as well.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3319
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1174
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 102
Illegal BsaI.rc site found at 743
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