Constitutive clostridial promoter Pcsp_fdx (BBa_K2715011), strong RBS and GFP reporter
Usage and Biology
Our project required the use of strong constitutive promoters which would function well in the non-model Gram-positive organism Clostridium difficile. We also wanted to establish whether these promoters functioned in E. coli, as this could have implications for cloning stages and vector assembly when trying to build constructs containing potentially toxic genes. In order to put the strength of these promoters into context, we decided upon a GFP fluorescence assay using the iGEM Interlab calibration curves, and also to compare their strengths to the Interlab positive and negative controls. This promoter was taken from the related species Clostridium sporogenes, driving expression of the ferredoxin gene, which has been shown in previous research to generally have strong expression in clostridial species.
In this composite part we've added a strong RBS BBa_K2715009, shown to function in Gram-positive and Gram-negative organisms, downstream of the BBa_K2715011 clostridial promoter, driving expression of GFP taken from BBa_E0040. The construct is part of a family of composite parts which all share the same strong RBS and GFP gene and were all characterised in the same plasmid backbone and in parallel in a fluorescence assay, the results of which can be seen below. The positive and negative controls are parts BBa_I20270 and BBa_R0040 respectively, used in the Interlab 2018 study.
The composite parts tested in this assay under the same conditions using a range of alternative promoters are as follows:
These composite parts were assembled in the shuttle cloning vector pMTL84151, and characterised in E. coli within this plasmid. Additionally these promoters were characterised in C. difficile using the gusA biobrick BBa_K330002 as a reporter gene in place of GFP, as GFP requires oxygen in order to function, and C. difficile is an anaerobic organism. The gusA containing composites used to assay the promoter activities in C. difficile are listed below.
The plasmid used for this characterisation in E. coli is displayed below.
This composite part has enabled a standardised characterisation of K2715011 when used in conjugation with a strong RBS shown to function in both Gram-positive and Gram-negative organisms, and its strength can be quantified in E. coli using the iGEM 2018 interlab units of fluorescence. The expression that was observed from this ferredoxin promoter in this GFP assay was strong, only superseded by the thiolase promoter and the iGEM Interlab positive control vector.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000Illegal BsaI.rc site found at 847
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.