Difference between revisions of "Part:BBa K3017067"
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<H2>Design</H2>
 
<H2>Design</H2>
 
<P>The asRNA characterization constructs all contain a constitutively expressed dCas9, a fluorescent protein RFP, asRNA under pBAD promoter, and sgRNA (GFP). 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 for GFP, suppression effect on the RFP should be non-existing. This allows us to assess the cross talking by sgRNA mismatch.</P>
 
<P>The asRNA characterization constructs all contain a constitutively expressed dCas9, a fluorescent protein RFP, asRNA under pBAD promoter, and sgRNA (GFP). 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 for GFP, suppression effect on the RFP should be non-existing. This allows us to assess the cross talking by sgRNA mismatch.</P>
 +
 +
<H3>Reference</H3>
 +
<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>
  
  

Revision as of 09:58, 20 October 2019


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

Aim

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

Design

The asRNA characterization constructs all contain a constitutively expressed dCas9, a fluorescent protein RFP, asRNA under pBAD promoter, and sgRNA (GFP). 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 for GFP, suppression effect on the RFP should be non-existing. This allows us to assess the cross talking by sgRNA mismatch.

Reference

Characterizing Genetic Circuit Components in E. coli towards a Campylobacter jejuni Biosensor

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

bioRxiv 290155; doi: https://doi.org/10.1101/290155


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