Part:BBa_K2715044
Constitutive expression of dCas9, and regulatory region of Toxin A in C. difficile driving GusA
Usage and Biology
This composite part is one component of a two plasmid system designed in order to assay the efficiency of binding of a range of synthetic guides in a CRISPRi experiment. The experiment involved having one composite part on one plasmid expressing a synthetic guide RNA complete with a unique targeting site for the Toxin A promoter of C. difficile. The other composite part is this module described here, and it contains a dcas9 coding sequence under constitutive expression of a strong clostridial promoter Pcac_thl, itself characterised in the following part BBa_K2715001. It also contains the regulatory region and promoter for Toxin A, driving expression of the reporter gene gusA, and has the alternative sigma factor required for activity of the promoter under control of the clostridial ferredoxin promoter, itself characterised in the following part BBa_K2715002. Each of these 3 modules are separated by a terminator sequence BBa_K2715014 , in order to represent read-through of the strong promoters into the native Toxin A promoter.
Characterisation
This composite part is one part of a two plasmid system, and as such is characterisation is linked to the unique guides which it facilitates the characterisation of, and these are all listed below.
These two plasmid hosts are part of the pMTL70000 series developed by the SBRC Nottingham, and possess two different replicons so that they can be maintained in the same strain of E. coli simultaneously. The respective strains were cultured over 10 hours from a normalised starting optical density, and a GusA assay performed to give an indication of the level of CRISPRi interference of each of the composite parts. In the case of a poorly performing guide, CRISPRi will be ineffective and levels of GusA activity will be similar to the negative control. In the case of a specific and well targeting guide, the promoter will be blocked by dcas9 binding, and expression of GusA will be reduced. This can be viewed as a good representation of the efficiency of each synthetic guide at controlling Toxin expression in the wild type organism C. difficile. The full characterisation of each of the guides can be found through their links above.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1515
Illegal XbaI site found at 4831
Illegal XbaI site found at 5047 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1515
Illegal NheI site found at 1274 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1515
Illegal BamHI site found at 3553 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1515
Illegal XbaI site found at 4831
Illegal XbaI site found at 5047 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1515
Illegal XbaI site found at 4831
Illegal XbaI site found at 5047 - 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.
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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