Part:BBa_K2715009

C.acetobutylicum thiolase gene RBS

Usage and Biology

This basic part is the ribosome binding site portion of the thiolase promoter from the Gram-positive organism Clostridium acetobutylicum. Thiolases, also known as acetyl-coenzyme A acetyltransferases (ACAT), are enzymes which convert two units of acetyl-CoA to acetoacetyl CoA in the mevalonate pathway, a key step in the core metabolism of C. acetobutylicum. This promoter and RBS has been demonstrated previously to be a strong promoter in clostridial species (ref).

Characterisation

This basic part was characterised as part of a composite part, and used as a strong RBS which functions in both the Gram-negative E. coli and the Gram-positive Clostridium difficile. The full native regulatory region driving thiolase expression in C. acetobutylicum is composed of the thiolase promoter BBa_K2715010, the 5' UTR BBa_K2715019 and this RBS BBa_K2715009. It was used to assess various promoter strengths in GFP assays and GUS assays, as outlined in the characterisation of the composite parts below.

GFP assay:

BBa_K2715106
BBa_K2715114
BBa_K2715119
BBa_K2715001
BBa_K2715002

GUS assay:

BBa_K2715025
BBa_K2715026
BBa_K2715027
BBa_K2715028
BBa_K2715029

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

Heap, J.T., Pennington, O.J., Cartman, S.T. and Minton, N.P., 2009. A modular system for Clostridium shuttle plasmids. Journal of microbiological methods, 78(1), pp.79-85.

Davis, D.F., Ward, W.W. and Cutler, M.W., 1994. Posttranslational chromophore formation in recombinant GFP from E. coli requires oxygen. In Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects. Proceedings of the 8th International Symposium on Bioluminescence and Chemiluminescence, Cambridge. Wiley, New York, NY (pp. 569-599).

Chiu, N.H. and Watson, A.L., 2017. Measuring β‐Galactosidase Activity in Gram‐Positive Bacteria Using a Whole‐Cell Assay with MUG as a Fluorescent Reporter. Current protocols in toxicology, 74(1), pp.4-44.