Part:BBa_K2992010

Synthetic guide RNA sequence 2 for botR integration

Synthetic guide RNA sequence 2 for the integration of several botR constructs at the pyrE locus of the C. sporogenes genome

Usage and Biology

Synthetic guide RNA sequence 2 for the use of CRISPR-Cas9 for genomic integration of various botR constructs in the generation of our volatile reporter strains for predicting neurotoxin production following food manufacture. Genomic integration of these constructs was at the pyrE locus of C. sporogenes.

Characterisation

This part was the guide RNA sequence that allowed our CRISPR/Cas9 system to work. This is the first of two alternative sgRNAs used for the genome-integration stage; we used two different sgRNAs in case one had a higher efficacy than the other. Upon integration of our botR - expression modules into the genome of C. spororgenes via CRISPR/Cas9 mediated repair, we replaced the pyrE gene with our botR- expression modules. This sgRNA guides the Cas9 protein to a specific site in the pyrE gene; any bacterium that has not undergone homologous recombination (a double crossover event that replaces pyrE with our botR expressing modules) will be cut by the Cas9 protein and consequentially be killed. The use of this part was used for the integration of parts BBa_K2992025, BBa_K2992026 and BBa_K2992027 in to the C. sporogenes genome. See results pagefor more

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

Raffestin, S., Dupuy, B., Marvaud, J. and Popoff, M. (2004). BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani. Molecular Microbiology, 55(1), pp.235-249.

Zhang, Z., Korkeala, H., Dahlsten, E., Sahala, E., Heap, J., Minton, N. and Lindström, M. (2013). Two-Component Signal Transduction System CBO0787/CBO0786 Represses Transcription from Botulinum Neurotoxin Promoters in Clostridium botulinum ATCC 3502. PLoS Pathogens, 9(3), p.e1003252.