Part:BBa_K2715022
C.difficile Toxin A promoter RBS
Usage and Biology
This basic part is the ribosome binding site portion of the tcdA toxin gene regulatory region from the Gram-positive organism Clostridium difficile. This promoter was taken directly from the organism in which we wished to demonstrate our novel methods (C. difficile) for controlling toxin production, as we wanted to gain a greater understanding of the level of expression from this promoter in the wild type organism. We decided to explore its expression levels in E. coli, as it has been shown in previous research to be under the regulation of an alternative sigma factor encoded by tcdR, present in the C. difficile genome, and therefore we expected to observe reduced expression in E. coli.
Characterisation
This basic part was characterised as part of a composite part, used to investigate the level of expression from this regulatory region in both E. coli and C. difficile. This composite part is composed of the native clostridial promoter driving expression of the toxin gene tcdA, sub-divided into the promoter region itself BBa_K2715012, the 5’ UTR region BBa_K2715021, and this RBS BBa_K2715022. 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 also assembled using gusA as a reporter gene, and this was used to assay its strength in Clostridium difficile. The composite part driving gusA is also listed below:
GFP assay:
BBa_K2715004
GUS assay:
BBa_K2715028
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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