Part:BBa_K3887004

pCas

pCas9 is a plamid containing Cas9 editing enzyme, used for gene modification at a specific location. Cas9 protein follows guide RNA to cut the two strands of DNA at a designated location, where strands of DNA can be edit (insertion, deletion,mutation). In our project，we used this plasmid to knockout TnaA on E. coli BL21(DE3) genome. Below is the map of it.

Figure.1 Snapgene map of pCas9

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 6015
  Illegal NotI site found at 3034
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 8294
  Illegal BamHI site found at 10296
  Illegal XhoI site found at 4353
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 10131
  Illegal AgeI site found at 11788
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 10113
  

Contribution: Tsinghua 2023 uses pCas to knock out ArgR gene

This year, Tsinghua 2023 successfully knocked out the ArgR gene in MG1655 using pTargetF/pCas. We adopted http://www.rgenome.net/cas-designer/notice Conduct N20 design. At the same time, we pioneered the simultaneous transformation of homologous arms, pCAS, and pTargetF into Escherichia coli, and successfully achieved gene knockout (see figure below). This innovation greatly reduces the operational complexity and experimental cycle of the prokaryotic gene knockout system.