Part:BBa_K2715010

Constitutive promoter from C.acetobutylicum thiolase gene

Usage and Biology

This basic part is the promoter portion of the thiolase regulatory region 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 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 promoter 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 this thiolase promoter BBa_K2715010, the 5' UTR BBa_K2715019 and the RBS BBa_K2715009. It's strength was assessed in E. coli using GFP as a reporter gene, the link to the characterisation data is provided below. A composite part was attempted to be assembled using gusA as a reporter gene, however this construct could not be cloned, likely due to the very high strength of the promoter in E. coli, and the fact that over expression of gusA is toxic to the cells. The attempted composite part driving gusA is also listed below:

GFP assay:

BBa_K2715001


GUS assay:

BBa_K2715025

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.