Part:BBa_K512002

Usage and Biology

Studies have shown that Escherichia coli bacteria have a system that provides immunity against viral infections and plasmid conjugation in the natural world. This recently discovered system, also known as the CRISPR/Cas system, is made of eight cas (CRISPR-associated) genes and an array of repeats and spacers known as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). The repeats are repeating DNA sequences of approximately thirty nucleotides that are separated by unique genetic elements, spacers. The sequences of these spacers are generally derived from foreign virus DNA or plasmids. The CRISPR array expands when the bacteria collects a new spacer in a process known as CRISPR adaptation. The bacteria only confers resistance to the virus or plasmid when its CRISPR repertoire contains a spacer that matches the DNA sequence of the invading DNA. Although parts of the pathway for CRISPR adaptation and CRISPR interference are being studied, many mechanisms such as spacer integration into the CRISPR array as well as the degradation signaling pathway for CRISPR-targeted DNA remain to be discovered.

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 cas3 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]

Such a system has been engineered by the Laboratory of Microbiology in the University of Wageningen and is displayed below:

K12 Inspired CRISPR cassette with alternating K12-targeting spacers and restriction sites

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 222
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 222
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 222
  Illegal BamHI site found at 344
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 222
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 222
  Illegal AgeI site found at 265
  
• 1000
  COMPATIBLE WITH RFC[1000]

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.