Difference between revisions of "Part:BBa K3017066"
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<partinfo>BBa_K3017066 short</partinfo>
 
<partinfo>BBa_K3017066 short</partinfo>
  
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<H2>Aim</H2>
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<P>The asRNA characterization construct is designed to prove asRNA ability to derepress a CRISPRi effect under arabinose induction with pBAD <i>in vivo</i>.</P>
 
<P>The asRNA characterization construct is designed to prove asRNA ability to derepress a CRISPRi effect under arabinose induction with pBAD <i>in vivo</i>.</P>
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<H2>Design</H2>
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<P>The asRNA characterization constructs all contain a constitutively expressed dCas9, a fluorescent protein GFP, 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). For all sgRNA transcription, weak Anderson promoter BBa_J23115 of strength 0.15 is used to allow for faster response rate between repressed state to derepressed state after arabinose induction.</P>
 
<P>The asRNA characterization constructs all contain a constitutively expressed dCas9, a fluorescent protein GFP, 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). For all sgRNA transcription, weak Anderson promoter BBa_J23115 of strength 0.15 is used to allow for faster response rate between repressed state to derepressed state after arabinose induction.</P>
  

Revision as of 00:24, 21 October 2019


Construct for testing CRISPRi asRNA de-suppression effect of CRISPRi sgRNA


The asRNA characterization construct is designed to prove asRNA ability to derepress a CRISPRi effect under arabinose induction with pBAD in vivo.


The asRNA characterization constructs all contain a constitutively expressed dCas9, a fluorescent protein GFP, 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). For all sgRNA transcription, weak Anderson promoter BBa_J23115 of strength 0.15 is used to allow for faster response rate between repressed state to derepressed state after arabinose induction.

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 3350
    Illegal PstI site found at 4772
    Illegal PstI site found at 4976
    Illegal PstI site found at 5006
    Illegal PstI site found at 6218
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 933
    Illegal NheI site found at 956
    Illegal NheI site found at 2326
    Illegal NheI site found at 2506
    Illegal NheI site found at 2529
    Illegal PstI site found at 3350
    Illegal PstI site found at 4772
    Illegal PstI site found at 4976
    Illegal PstI site found at 5006
    Illegal PstI site found at 6218
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2811
    Illegal BamHI site found at 2265
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 3350
    Illegal PstI site found at 4772
    Illegal PstI site found at 4976
    Illegal PstI site found at 5006
    Illegal PstI site found at 6218
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 3350
    Illegal PstI site found at 4772
    Illegal PstI site found at 4976
    Illegal PstI site found at 5006
    Illegal PstI site found at 6218
    Illegal NgoMIV site found at 3638
    Illegal NgoMIV site found at 4742
    Illegal NgoMIV site found at 4815
    Illegal NgoMIV site found at 5300
    Illegal NgoMIV site found at 6209
    Illegal AgeI site found at 2100
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 705
    Illegal SapI site found at 2082