Part:BBa_K1026002

Constitutively Expressed gRNA targeting mRFP

This Constitutively Expressed gRNA targeting mRFP is a no-BioBrick-scars assembly of the constitutive promoter BBa_J23100, DNA sequence of a gRNA that targets mRFP reporter and a reliable terminator BBa_B0015.

This gRNA sequence comes from the following literature: 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.

In CRISPRi system, when dCas9 and gRNA bind together, they will bind to the specific DNA that has the complementary sequence of gRNA. 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.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]