Part:BBa_K3595014

DNA that forms a guide RNA to guide cas9 to cut the gene

Cas9 protein in CRISPR-cas9 needs a guide RNA to combine with so that it can cut the certain position on the DNA. In knocking out the argR gene, the guide RNA is transcripted by the loaded DNA.

Usage and Biology

This sequence of DNA is used to help cas9 protein to cut the specific position on the DNA. It is constructed by connecting pUC, Ampr, RBS J23119, pTarget-upArm-gRNA-downarm. It is induced by the arabinose.

Experimental Setup

• Transform the plasmid pTarget with up-arm, down-arm, and gRNA to the competentE.coli DH10b from company transgene.
• Transform the plasmid pCas into the competent E.coli with previous plasmid mentioned.
• Add arabinose
• Cultivate the E.coli with the temperature at 30 degree Celsius overnight.
• Use colony PCR to test if the gene is successfully knocked out.

Results

• As shown on the graph, the argR is knocked out with the decrease of 500bp which is the length of argR.

Contrast of E.coli DH10b genome before and after the deletion of argR. (A)DH10b with argR. (B) DH10b genome without argR. (C) The electrophoresis graph of DH10b genome with and without argR. Line 1 is marker (RB-MKS); Line 2 is wildtype; Line 3 is DH10b ∆argR

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]