Part:BBa_K1026000

Constitutively Expressed dCas9 Operon

This Constitutively Expressed dCas9 Operon is an assembly of the constitutive promoter BBa_J23100, a strong RBS BBa_B0034, coding sequence of dCas9 protein and a reliable terminator BBa_B0015. dCas9 is a mutant of Cas9, with its endonuclease activity wiped out. dCas9 acts as the sole protein in CRISPRi system (Qi et al., 2013).

Experiment

In CRISPRi system, when dCas9 and gRNA bind together, they will bind to the specific DNA and block the RNA Polymerase, thus knocking down that gene. We test this system using mRFP as a reporter gene.

There are two experimenting groups: e.coli in control group only expresses dCas9 and mRFP, and in case group it expresses dCas9, sgRNA and mRFP. And the result is shown as follows:

In case group, dCas9 did not affect the expression of mRFP; in control group, there is a significant knock down effect of mRFP.

This result is in consistent with our expectation.

Reference

QI, LEI S., LARSON, MATTHEW H., GILBERT, LUKE A., DOUDNA, JENNIFER A., WEISSMAN, JONATHAN S., ARKIN, ADAM P. & LIM, WENDELL A. 2013. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 152, 1173-1183.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1401
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1401
  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 1160
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1401
  Illegal BamHI site found at 3439
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1401
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1401
  
• 1000
  COMPATIBLE WITH RFC[1000]