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 an alternative RNA sequence that allowed our CRISPR system to work. As we integrated BotR in to the genome via double homologous crossover (replacing the pyrE gene), the CRISPR systems was designed to kill the C. spororgenes that botR did not successfully integrate in to, this part was the alternative guide RNA sequence that allowed the CRISPR system to cut in the event a double homologous crossover did not happen. The use of this part made the integration of parts BBa_K2992025, BBa_K2992026 and BBa_K2992027 in to the C. sporogenes genome possible.

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.