Difference between revisions of "Part:BBa K3017066"
 
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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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 +
<p>asRNA is a purely synthetic RNA that is designed to bind with its paired sgRNA at its extensor region. By removing the secondary structure of sgRNA extensor, dCas9 leave the sgRNA. The CRISPRi repression is reversed by the asRNA.[1]</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>
 
<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>
  
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<H3>Reference</H3>
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<P>Characterizing Genetic Circuit Components in E. coli towards a Campylobacter jejuni Biosensor</P>
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<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>
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<br>
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<P>bioRxiv 290155; doi: https://doi.org/10.1101/290155</P>
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<p><b>References:</b></p>
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<br>[1] Y. J. Lee, A. Hoynes-Oconnor, M. C. Leong, and T. S. Moon, “Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system,” Nucleic Acids Research, vol. 44, no. 5, pp. 2462–2473, Feb. 2016.
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<br>[2] C. Anders, O. Niewoehner, A. Duerst, and M. Jinek, “Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease,” Nature, vol. 513, no. 7519, pp. 569–573, 2014.
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<br>[3] S. H. Sternberg, S. Redding, M. Jinek, E. C. Greene, and J. A. Doudna, “DNA Interrogation by the CRISPR RNA-Guided Endonuclease Cas9,” Biophysical Journal, vol. 106, no. 2, 2014.
 +
<br>[4] T. Karvelis, G. Gasiunas, A. Miksys, R. Barrangou, P. Horvath, and V. Siksnys, “crRNA and tracrRNA guide Cas9-mediated DNA interference inStreptococcus thermophilus,” RNA Biology, vol. 10, no. 5, pp. 841–851, 2013.
 +
<br>[5] T. Møller, T. Franch, P. Højrup, D. R. Keene, H. P. Bächinger, R. G. Brennan, and P. Valentin-Hansen, “Hfq,” Molecular Cell, vol. 9, no. 1, pp. 23–30, 2002.
 +
<br>[6] G. M. Cech, A. Szalewska-Pałasz, K. Kubiak, A. Malabirade, W. Grange, V. Arluison, and G. Węgrzyn, “The Escherichia Coli Hfq Protein: An Unattended DNA-Transactions Regulator,” Frontiers in Molecular Biosciences, vol. 3, 2016.
 +
<br>[7]N. Brzozowska et al., “Characterizing Genetic Circuit Components in E. coli towards a Campylobacter jejuni Biosensor,” p. 290155, 2018.
  
  

Latest revision as of 01:55, 22 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.

asRNA is a purely synthetic RNA that is designed to bind with its paired sgRNA at its extensor region. By removing the secondary structure of sgRNA extensor, dCas9 leave the sgRNA. The CRISPRi repression is reversed by the asRNA.[1]


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.



References:


[1] Y. J. Lee, A. Hoynes-Oconnor, M. C. Leong, and T. S. Moon, “Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system,” Nucleic Acids Research, vol. 44, no. 5, pp. 2462–2473, Feb. 2016.
[2] C. Anders, O. Niewoehner, A. Duerst, and M. Jinek, “Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease,” Nature, vol. 513, no. 7519, pp. 569–573, 2014.
[3] S. H. Sternberg, S. Redding, M. Jinek, E. C. Greene, and J. A. Doudna, “DNA Interrogation by the CRISPR RNA-Guided Endonuclease Cas9,” Biophysical Journal, vol. 106, no. 2, 2014.
[4] T. Karvelis, G. Gasiunas, A. Miksys, R. Barrangou, P. Horvath, and V. Siksnys, “crRNA and tracrRNA guide Cas9-mediated DNA interference inStreptococcus thermophilus,” RNA Biology, vol. 10, no. 5, pp. 841–851, 2013.
[5] T. Møller, T. Franch, P. Højrup, D. R. Keene, H. P. Bächinger, R. G. Brennan, and P. Valentin-Hansen, “Hfq,” Molecular Cell, vol. 9, no. 1, pp. 23–30, 2002.
[6] G. M. Cech, A. Szalewska-Pałasz, K. Kubiak, A. Malabirade, W. Grange, V. Arluison, and G. Węgrzyn, “The Escherichia Coli Hfq Protein: An Unattended DNA-Transactions Regulator,” Frontiers in Molecular Biosciences, vol. 3, 2016.
[7]N. Brzozowska et al., “Characterizing Genetic Circuit Components in E. coli towards a Campylobacter jejuni Biosensor,” p. 290155, 2018.


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