Difference between revisions of "Part:BBa K3081024"
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This composite part is the principal design of the inducible CRISPRi system, with assistance of constantly expressed sgRNA (NT1) that is complementary to the corresponding sequence of mRFP coding region. Fluorescence is decreased as the concentration of arabinose increases.
 
This composite part is the principal design of the inducible CRISPRi system, with assistance of constantly expressed sgRNA (NT1) that is complementary to the corresponding sequence of mRFP coding region. Fluorescence is decreased as the concentration of arabinose increases.
  
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To investigate CRISPR-dCas9 binding specificity and affinity with DNA, we constructed arabinose-induced expression of dCas9 targeted to mRFP coding region, with assistance of constantly-expressed single guide RNA that is complementary to the corresponding sequence. Since normal mRNA elongation is interrupted by occurrence of dCas9, fluorescence is greatly decreased as the arabinose concentration increases, comparing to a single guide RNA that has no binding specificity to DNA. This proves the basic concept that a dCas9 protein is able to bind to DNA with sequence specificity and interferes with the physiological process.
  
  

Revision as of 09:07, 21 October 2019


pBAD-dCas9-J23119-NT1-J23119-mRFP

This composite part is the principal design of the inducible CRISPRi system, with assistance of constantly expressed sgRNA (NT1) that is complementary to the corresponding sequence of mRFP coding region. Fluorescence is decreased as the concentration of arabinose increases.

To investigate CRISPR-dCas9 binding specificity and affinity with DNA, we constructed arabinose-induced expression of dCas9 targeted to mRFP coding region, with assistance of constantly-expressed single guide RNA that is complementary to the corresponding sequence. Since normal mRNA elongation is interrupted by occurrence of dCas9, fluorescence is greatly decreased as the arabinose concentration increases, comparing to a single guide RNA that has no binding specificity to DNA. This proves the basic concept that a dCas9 protein is able to bind to DNA with sequence specificity and interferes with the physiological process.


T--Peking--CRISPRi_ssrA_ctrl_NT1.png

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1470
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
    Illegal AgeI site found at 6173
    Illegal AgeI site found at 6285
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961