Part:BBa_K2715006:Design
E.coli promoter BBa_J23119 driving synthetic guide 2 targeting tcdB promoter
- 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]
Design Notes
This is a composite part constructed to express a synthetic guide RNA designed to work with the dCas9 protein from S. pyrogenes. It is composed of the previously used constitutive E. coli promoter BBa_J23119, a unique 20 bp guide targeting sequence BBa_K2715033, a synthetic guide RNA for S. pyrogenes dCas9 protein BBa_K2715042, and a terminator sequence BBa_K2284000.
Source
The promoter J23119 is present in pSB1A2 and sourced from the iGEM registry, the unique guide sequence is sourced from the promoter region of toxin A from C. difficile. The synthetic guide RNA sequence is sourced from the CRISPR vector described in CRISPR/Cas9-based efficient genome editing in Clostridium ljungdahlii, an autotrophic gas-fermenting bacterium. ACS synthetic biology, 5(12), pp.1355-1361 (see refs below). The terminator is sourced from the pMTL80000 modular vector series developed by the SBRC Nottingham (see refs below).
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.
Huang, H., Chai, C., Li, N., Rowe, P., Minton, N.P., Yang, S., Jiang, W. and Gu, Y., 2016. CRISPR/Cas9-based efficient genome editing in Clostridium ljungdahlii, an autotrophic gas-fermenting bacterium. ACS synthetic biology, 5(12), pp.1355-1361.