Part:BBa_K512001

casABCDE12

Usage and Biology

casABCDE12 is a series of cas (CRISPR-associated) genes responsible for CRISPR interference in E. coli. These genes make up seven of the eight cas genes involved in the assembly of the Cascade (CRISPR-associated complex for antiviral defence) complex. This complex has been studied to provide immunity to viral infections and plasmid conjugation. casE, in particular, has been studied to be an endonuclease in E. coli that cleaves precursor CRISPR RNA into small crRNAs that bind with Cascade and guide the defense against foreign viruses and plasmids.

Additional studies have shown that only cas genes casABCDE and cas3 are required for CRISPR interference to work.

Proof of operation:

In the chart above, BL-D1 are the cells without IPTG, BL-D2 are with IPTG; and for BL-D3, we add IPTG 2 hours later. IPTG can induce CRISPR to target the plasmid. As we can see from the chart, with casABCDE genes and CRISPR, the growth rate and optical density are decreased significantly compare to the cells without IPTG.

For complete and other results, please check the link [http://2011.igem.org/Team:USC/Project]

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 84
  Illegal PstI site found at 1253
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 84
  Illegal PstI site found at 1253
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3094
  Illegal BglII site found at 4279
  Illegal BamHI site found at 23
  Illegal BamHI site found at 3224
  Illegal BamHI site found at 3585
  Illegal BamHI site found at 4482
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 84
  Illegal PstI site found at 1253
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 84
  Illegal PstI site found at 1253
  Illegal NgoMIV site found at 5203
  Illegal AgeI site found at 784
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1764
  

Source

casABCDE12 has been cloned from genomic DNA of DH5a.

References

1. Brouns, S.J.J., et. al. (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321: 960-964.
2. Westra, E.R., et. al. (2010) H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO. Molecular Microbiology 77:1380-1393.