Composite

Part:BBa_K3081024

Designed by: Le Zhang   Group: iGEM19_Peking   (2019-10-16)
Revision as of 19:14, 21 October 2019 by SyqOVO (Talk | contribs)

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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
Figure 1. Comparison between dCas9 and dCas9-ssrA system by CRISPRi effect on mRFP fluorescence.


Reference:

[1]Wolański M, Donczew R, Zawilakpawlik A, et al. oriC-encoded instructions for the initiation of bacterial chromosome replication.[J]. Frontiers in Microbiology, 2015, 5(735):735.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
    Illegal NheI site found at 5459
    Illegal NheI site found at 5482
    Illegal NheI site found at 5590
    Illegal NheI site found at 5613
  • 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


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Categories
Parameters
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