Part:BBa_K1075026

AraC-pBad-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 E.coli(ec)ssrA(DAS+4) tag and a double terminator.

biology

AraC-pbad is an arabinose inducible regulatory promoter/repressor unit.

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 proteins 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 2726
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961