Part:BBa_K2715039
E.coli promoter BBa_J23119 driving synthetic guide 4 targeting tcdB promoter
Usage and Biology
This is a composite part constructed to express a synthetic guide RNA designed to work with the dCas9 protein from S. pyrogenes. It is specific to a portion of the promoter region controlling expression of the Toxin A gene in C. difficile by virtue of the unique 20bp guide targeting sequence, in this case BBa_K2715035. It is also flanked with a terminator to prevent read through expression into the plasmid backbone. In our project we aimed to use a nucleolytically inactive Cas9 protein (dCas9) in combination with this unique guide RNA sequence to block the transcription machinery from being able to transcribe tcdA in C. difficile, thus reducing its toxicity. 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 BBa_K2715044 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.
Multiple guide sequences were designed so that their relative targeting efficiencies could be compared in an assay. The complete list of guides designed can be found below.
Characterisation
In order to test whether this composite part is effective in being able to reduce expression of the tcdA promoter, we designed an assay using the reporter GusA in place of the C. difficile toxin A.
This composite part is one part of a two plasmid system, and its characterisation was performed in parallel with a range of six unique guides, and these are all listed below. The other composite required for all these examples is BBa_K2715044.
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. Two alternative plasmid host strains were used for expression of the synthetic guide composite part, one with a kanamycin resistance cassette, and one with erythromycin. Unfortunately two of the synthetic guides, number 2 and number 6, could not be cloned in the kanamycin plasmid backbone, but all six guides were able to be cloned in the erythromycin resistant plasmid. The composite part containing dcas9 and gusA regulatory components BBa_K2715044 was maintained in a plasmid containing a chloramphenicol resistance marker.
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 results of the assay are displayed below, with analysis of OD normalised and non-normalised data provided:
Repression efficiency of different sgRNAs against the PCdi_tcdA promoter
Repression efficiency of sgRNA variants (A1-6) was measured using a β-Glucuronidase activity assay in E. coli strains harbouring two plasmids containing the two required composite parts. Briefly, strains were grown in media with the necessary antibiotics and overnight cultures (A, C) or cultures at the mid-log growth phase [OD600 ≈ 1.0] (B, D) were harvested by centrifugation. Subsequently, cell pellets were resuspended in 500 μL of a suitable buffer and 75 μL of the cell suspension were reacted with 28.4 μM of 4-methylumbelliferyl-β-D-glucuronide. Fluorescence intensity was monitored over a period of 10 min at 440-460 nm using an excitation wavelength of 355-375 nm. Data represent mean values of three technical replicates ± SD. Statistical analysis was carried out using one-way ANOVA with Dunnett’s test for multiple comparisons against the control strain (c); p-values are indicated as: 0.1234 (ns), 0.0332 (*), 0.0002 (***), <0.0001 (****).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 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.
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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