Part:BBa_K3875022

Usage and Biology

22: J23119 promoter- gRNA targeting fadR- Cas9 binding- Terminator
Introduction
To explain this part clearly, we would like to introduce the background knowledge of CRIPSR/Cas9 gene-editing system. This technology comes from bacterial immune system. The bacteria will use this system to cut the DNA and store a part of the sequence in the host genome, if phages or plasmids get into the cell. Nowadays, we use this tech to cut the specific DNA in genetic engineering. The CRISPR/Cas9 system has attracted attention in recent years for its ability to edit and regulate DNA in a wide variety of organisms and cell types. Using a strand of single guide RNA (sgRNA), the Cas9 protein is able to search a cellular genome and induce double stranded breaks at a target sequence complementary to the sgRNA that can then be modified. The sgRNA molecules co-expressed with Cas9 each consist of three segments: a 20 nucleotide (nt) target-specific complementary region, a 42 nt Cas9-binding hairpin (Cas9 handle), and a 40 nt transcription terminator.
In order to cut the DNA efficiently, we need two kinds of plasmid. One is pCas9 which products the Cas9 protein, the other is pTarget which will transcribe the sgRNA. Once in the recipient cell, pCas9 expressed Cas9 endonuclease and crRNA or gRNA that cut a target DNA on the pTarget. DNA cut led to degradation of the pTarget. Our project wants to enhance the β-oxidation in E.coli nissle 1917, so we cut the fadR which represses the expression of above 15 genes. The N20 sequence is 5’-GGAATAACCGCTTCCCTCCC-3’. This sequence will help the Cas9 protein and gRNA complex to locate the knockout site. When the Cas9 protein finds this target sequence with the PAM, the protein will melt the bases immediately upstream of the PAM and pair them with the complementary region on the guide RNA. If the complementary region and the target region pair properly, the RuvC and HNH nuclease domains will cut the target DNA after the third nucleotide base upstream of the PAM.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 30
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal SpeI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 30
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 30
  
• 1000
  COMPATIBLE WITH RFC[1000]