Difference between revisions of "Part:BBa K2715011"
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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*10<sup>3</sup> MEFL/particle and 1.1*10<sup>6</sup> MEFL/particle. | 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*10<sup>3</sup> MEFL/particle and 1.1*10<sup>6</sup> MEFL/particle. | ||
Revision as of 10:55, 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 <a href="https://2019.igem.org/Team:Nottingham/Results">Results</a> page.
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.