Part:BBa_K1100152

Csy4-16nt interaction estimation device

In this device, we test the interaction between csy4 and its 16-nt cleavage site in vivo.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 67
  Illegal SpeI site found at 885
  Illegal PstI site found at 1775
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 67
  Illegal SpeI site found at 885
  Illegal PstI site found at 1775
  Illegal NotI site found at 73
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 67
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 67
  Illegal SpeI site found at 885
  Illegal PstI site found at 1775
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 67
  Illegal XbaI site found at 82
  Illegal SpeI site found at 885
  Illegal PstI site found at 1775
  Illegal NgoMIV site found at 536
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 252
  

Background

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Csy4 is a member of CRISPR pathway discovered in Pseudomonas aeruginosa. It is a single endoRNase that recognizes and cleaves a 28-nucleotide repetitive sequence and produces stable transcripts with a 5’hydroxylgroup, which can eliminate unwanted interactions between 5’UTRs and translational elements such as RBSs units to standardize the expression of the elements. ( Lei Qi et al, 2012).The cleavage site can be curtailed to 16nt proved by in vitro data (Rachel E. Huarwitz et al, 2010)

The CRISPR RNA-processing system facilitate engineering of standard genetic elements in various contexts( Lei Qi et al, 2012).

Figure. 1. The CRISPR RNA-processing system facilitate engineering of standard genetic elements in various contexts( Lei Qi et al, 2012).

Design

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We introduced the csy4 platform and its cleavage platform onto one plasmid as followed: pSB1C3-BBa_R0040-16nt-BBa_B0034-Csy4-BBa_B0015-BBa_R0063-16nt-BBa_B0034-sfGFP, in order to let the csy4 express to recognize and cleave the 16-nucleotide sequence.

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Reference:

Haurwitz R E, Jinek M, Wiedenheft B, et al. Sequence-and structure-specific RNA processing by a CRISPR endonuclease[J]. Science, 2010, 329(5997): 1355-1358. Qi L, Haurwitz R E, Shao W, et al. RNA processing enables predictable programming of gene expression[J]. Nature biotechnology, 2012, 30(10): 1002-1006. Cambray G, Guimaraes J C, Mutalik V K, et al. Measurement and modeling of intrinsic transcription terminators[J]. Nucleic acids research, 2013, 41(9): 5139-5148.