Composite

Part:BBa_K3017068

Designed by: Luk Hau Ching   Group: iGEM19_Hong_Kong_HKUST   (2019-10-17)
Revision as of 00:29, 21 October 2019 by HannahLuk (Talk | contribs)


Construct for testing CRISPRi asRNA cross-talk with non-target sgRNA


This asRNA characterization construct is to characterize the cross talking by asRNA mismatch with the incorrect sgRNA.


This asRNA characterization constructs all contain a constitutively expressed dCas9, a fluorescent protein RFP, asRNA (for sgRNA_GFP) under pBAD promoter, and sgRNA (RFP). Under the regulation of pBAD promoter, asRNA is transcripted when arabinose is added to the culture medium. The transcription start site of the pBAD promoter has been identified according to Brzozowska et al. (2018), in which the asRNA has been placed on the Transcription Start Site (TSS). Forl sgRNA transcription, weak Anderson promoter BBa_J23115 of strength 0.15 is used.

Since the sgRNA transcribed will be targeting for rfp, suppression effect on the RFP should be observed. A matching asRNA should reverse the suppression. Therefore, upon L-arabinose induction for the transcription of asRNA, no derepression effect is expected to be observed as the asRNA’s extensor is not complementary to the sgRNA_RFP.</P>

Reference

<P>Characterizing Genetic Circuit Components in E. coli towards a Campylobacter jejuni Biosensor</P> <P>Natalia Brzozowska, Jane Gourlay, Ailish O’Sullivan, Frazer Buchanan, Ross Hannah, Alison Stewart, Hannah Taylor, Reuben Docea, Greig McLay, Ambra Giuliano, James Provan, Katherine Baker, Jumai Abioye, Julien Reboud, Sean Colloms</P> <P>bioRxiv 290155; doi: https://doi.org/10.1101/290155</P>


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 3336
    Illegal PstI site found at 4758
    Illegal PstI site found at 4962
    Illegal PstI site found at 4992
    Illegal PstI site found at 6204
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 919
    Illegal NheI site found at 942
    Illegal NheI site found at 2312
    Illegal NheI site found at 2492
    Illegal NheI site found at 2515
    Illegal PstI site found at 3336
    Illegal PstI site found at 4758
    Illegal PstI site found at 4962
    Illegal PstI site found at 4992
    Illegal PstI site found at 6204
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2797
    Illegal BamHI site found at 2251
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 3336
    Illegal PstI site found at 4758
    Illegal PstI site found at 4962
    Illegal PstI site found at 4992
    Illegal PstI site found at 6204
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 3336
    Illegal PstI site found at 4758
    Illegal PstI site found at 4962
    Illegal PstI site found at 4992
    Illegal PstI site found at 6204
    Illegal NgoMIV site found at 3624
    Illegal NgoMIV site found at 4728
    Illegal NgoMIV site found at 4801
    Illegal NgoMIV site found at 5286
    Illegal NgoMIV site found at 6195
    Illegal AgeI site found at 616
    Illegal AgeI site found at 728
    Illegal AgeI site found at 2086
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 2068

[edit]
Categories
Parameters
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