Translational_Unit
Csy4-Y176F

Part:BBa_K2615005:Design

Designed by: Yunqian Zhang   Group: iGEM18_OUC-China   (2018-09-06)
Revision as of 12:34, 11 September 2018 by Anyi (Talk | contribs) (→‎Design Notes)


Csy4-Y176F, the No.3 member of Csy4 family.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 377
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 93


Design Notes

We designed this part by point mutation. We changed the TAC(encoding Tyr) to TTT(encoding Phe) on the 176th site based on wild type Csy4.

Source

The part is designed by OUC-China. The wild type Csy4 existed in Pseudomonas aeruginosa. In Pseudomonas aeruginosa, crRNA biogenesis requires the endoribonuclease Csy4, which binds and cleaves the repetitive sequence of the CRISPR transcript. Biochemical assays and three co-crystal structures of wild-type and mutant Csy4/RNA complexes reveal a substrate positioning and cleavage mechanism in which a histidine deprotonates the ribosyl 20-hydroxyl pinned in place by a serine, leading to nucleophilic attack on the scissile phosphate.

References