Difference between revisions of "Part:BBa K1100152"
 
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<partinfo>BBa_K1100152 short</partinfo>
 
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In this device, we test the interaction between csy4 and its 16-nt cleavage site ''in vivo'' to be a supplementary experiment for the Rachel E. Huarwitz et al, and Lei Qi et al's previous work.
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In this device, we tested the interaction between csy4 and its 16-nt cleavage site ''in vivo'' to be a supplementary experiment for the Rachel E. Huarwitz et al, and Lei Qi et al's previous work.
  
 
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Latest revision as of 00:49, 28 September 2013

Csy4-16nt interaction estimation device

In this device, we tested the interaction between csy4 and its 16-nt cleavage site in vivo to be a supplementary experiment for the Rachel E. Huarwitz et al, and Lei Qi et al's previous work.

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

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

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.

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.