Part:BBa_K2754000

dCas9 regulated by a TetR inducable promoter

This part includes the coding sequence for dead Cas9 regulated by a tetracycline inducable promoter (tetR/tetA promoter), a strong RBS and two terminators (rrnB T1 terminator and T7Te terminator). In addition, it contains an upstream TetR sequence which is also controlled by the tetR/tetA promoter.

Note: The characterization of this part was done before making it completely RFC10 compatible. It is demonstrated to work before making it RFC10 compatible.

Please remember that this dCas9 part must be used in combination with some sort of gRNA encoding part, for example the plasmid from https://www.addgene.org/44251/.

Below is a graph of results when the dCas9 is targeted to the LuxS gene, which is involved in biofilm production in E. coli . When dCas9 and gRNA is expressed to create a functional CRISPRi system, it shows here that biofilm production is reduced in E. coli TGI, which indicates that the dCas9 is working (again keep in mind that this is before making the dCas9 part RFC10 compatible).

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Figure 1 : Absorbance of accumulated biofilm measured in Crystal Violet Assay as a function of incubation time of the biofilm. Biofilm from E. coli TGI cells containing the dCas9 part (before making it RFC10 compatible) and a gRNA part were tested here. A clear reduction of accumulated biofilm is displayed

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Figure 2: Vector map of the RFC10 compatible dCas9 inserted in pBS1C3 with annotations. Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1832
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 710
  Illegal BamHI site found at 4111
  Illegal XhoI site found at 4841
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]