Difference between revisions of "Part:BBa K1075027"

Revision as of 17:48, 3 October 2013

SplitSspB(Rapamycin inducable)-pLac-RBS34-mCherry-(Ec)ssrA(DAS+4)-TT

a

construction

The part contains the promoter plac, a ribosomal binding site, the red fluorescent protein mCherry fused to the ecssrA (DAS+4) tag and a double terminator. It is integrated in the plasmid pJD427, which contains a SspB split system.

biology

The plasmid pJD427 contains the two parts of splitted SspB, 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 ssrA 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 proteins SspB binding and delivering substrates to the proteases in order to make the process more efficient. The mutated ssrA (DAS+4) weakens a direct binding between proteases and ssrA and increases the dependance of sspB. [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 SspB, we tag the red fluorescent protein mCherry with ssrA (DAS+4) to measure the degradation rate. We control SspB 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.

Sequence and Features