Part:BBa_K3081032

pBAD-dCas9-ssrA-J23119-R1+(18bp)

This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, with the 18 bp sgRNA targeting to the R1+ DnaA box on E.coli genome replication initiation region, OriC. In natural situations, R1+ is a high affinity box for DnaA binding. By blocking the binding of DnaA protein to R1+ box using a 18bp sgRNA, alleviation of severe arrest and inhibition to the genome replication initiation is achieved. This part is an improvement of BBa_K3081009, which we add a degradation signal peptide ssrA to the dCas9. This largely accelerates the degradation rate of dCas9 and weakens its overinhibtion on genome replication initiation targeted to the R1 box.

Our experiments reveal that multi-input control is achievable. The variables include length of sgRNA. We observed corresponding changes in cell morphology (cell elongation in most cases) among the 18 bp, 19bp, and 20bp sgRNA targeting to the R1+ DnaA box on E.coli genome replication initiation region, OriC.

R1+ssrA 20bp

R1+ssrA 19bp

R1+ssrA 18bp

（Considering the technical problem, if the reader is interested in our results, welcome to our presentation and poster session to watch the original movie.）

Reference：

[1]Wolański M, Donczew R, Zawilakpawlik A, et al. oriC-encoded instructions for the initiation of bacterial chromosome replication.[J]. Frontiers in Microbiology, 2015, 5(735):735.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  Illegal NheI site found at 5459
  Illegal NheI site found at 5482
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1470
  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961