Part:BBa_K1075025

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.

biology

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.

Application as a bacterial fotographic film might be possible as well.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1717
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]