Part:BBa_K1323002

dCas9: Expression cassette under a xylose inducible promoter

dCas9 or ‘dead’ cas9 is a mutated version of Cas9 that has lost its endonuclease activity - it no longer cuts double stranded DNA, instead dCas9 simply binds to it. This means dCas9 can function as a repressor after complexing with sgRNA (BBa_K1323000, BBa_K1323001) and the sgRNA-target site. dCas9 contains mutations in the RuvC1 and HNH nuclease domains; these changes are what bestow this modified Cas9 with its ability to bind DNA instead of cleaving it (Qi et al., 2013). BBa_K1323002 has a Xylose Inducible Promoter BBa_K1323014 and a sodA RBS BBa_K1323022. The illegal sites have been removed, and the sequence was codon optimized for expression in S.epidermidis using JCat software.

This part was DNA-synthesized by Bio Basic.

Usage and Biology

Contribution
Section below is the contribution Team Tsinghua made to this part. We characterized this part by transforming the part into E. coli and validated its sequence. In addition, in order to perform imaging related experiments, we fused a green fluorescence protein to this construct. As is indicated below, the nuclear localization of dCas9 protein can be well visualized.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1476
  Illegal BamHI site found at 4894
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

Qi, L.S. et al., (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 1173-1183.