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
Group: Team Tsinghua 2016.
Author: Tianyang Mao.
Summary: Section below is the contribution Team Tsinghua made to previous parts . We characterized three parts, one of which belongs to this page (https://parts.igem.org/Part:BBa_K1323002, https://parts.igem.org/Part:BBa_K1493504 and https://parts.igem.org/Part:BBa_K1470002) by transforming the part into E. coli and validated its sequence using enzymatic digestion as well as sequencing. We functionally improved these parts by fusing three parts together (dCas9 from BBa_K1323002, GFP from BBa_K1493504, and Gal4BD from BBa_K1470002). In a sentence, the nuclear localization of dCas9 protein can be well visualized. More controlled experiments suggest we successfully repurposed CRISPR/Cas9 for transcriptional control of an exogenous suicidal system contingent upon the fidelity of canonical mRNA sequences. For detailed documentation, please refer to the part page https://parts.igem.org/Part:BBa_K1923011.

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.