Difference between revisions of "Part:BBa K2715011"

Revision as of 10:56, 21 October 2019

Constitutive promoter from C.sporogenes ferrodoxin gene

Usage and Biology

This basic part is the promoter portion of the ferredoxin regulatory region from the Gram-positive organism Clostridium sporogenes. Ferredoxins (from Latin ferrum: iron + redox, often abbreviated "fdx") are iron–sulfur proteins that mediate electron transfer in a range of metabolic reactions, and the ferredoxin gene is known is to highly expressed in C. sporogenes. 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. sporogenes is composed of this ferredoxin promoter BBa_K2715011 and it's RBS BBa_K2715020. 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_K2715002


GUS assay:

BBa_K2715026


The first observation from the expression of the FAST protein using different clostridium and E. coli promoters is that FAST is a suitable reporter gene, both in E. coli and in Clostridium Sporogenes. Indeed, quantifiable levels of fluorescence were recorded in between 6.3*10³ MEFL/particle and 1.1*10⁶ MEFL/particle.

Pfdx and Bba_K2715011 have equivalent expression levels (5.5*10⁵ +- 0.9*10⁵ MEFL/particle and 6*10⁵ +-1*10⁵ MEFL/particle respectively in C. sporogenes; 9*10⁵ +- 1*10⁵ 10⁵ MEFL/particle and 1.007*10⁶ +-0.009*10⁶ 10⁵ MEFL/particle respectively in E. coli). As such, Pfdx and Bba_K2715011 are interchangeable. However, since our part “Pfdx” is the original, unmutated version of the ferredoxin promoter from C. sporogenes, the sequence of Bba_K2715011 documented in the registry of parts should be curated to match the native sequence of Pfdx.</p>

For more characterisation details, please see the Results page (https://2019.igem.org/Team:Nottingham/Results).

Sequence and Features

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.