Difference between revisions of "Part:BBa K2381012"
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<partinfo>BBa_K2381012 short</partinfo>  
 
<partinfo>BBa_K2381012 short</partinfo>  
  
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It’s a gRNA plasmid which helps our CRISPR-System target to the MG1655 OriC sequence in order to achieve our expectation
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this year. Its backbone is taken from iGEM Kit Plate named BBa_K165005, but the BsaI restriction site which was in Amp.  
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coding sequence has been deleted and a synonymous mutation has occurred by overlapping PCR. We used J23119 promoter, a
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high-intensity promoter, to obtain a higher expression of gRNA than dCas9. The 21bp-sequence comes after is a kind of
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It’s a gRNA sequence containing 20bp targeting at <i>oriC</i> and a full-length gRNA scaffold. The sequence is central to our project and is related with all the system built this year.<br/>
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sequence types taken from the article that is complementary to the Oric sequence and has a nice effect on our project. The
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The targeting sequence of this part is acquired from reference paper<sup>1</sup>.  
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full length gRNA scaffold is also designed after consulting literatures.
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<img src="https://static.igem.org/mediawiki/parts/d/d2/T--HZAU-China--gRNA_function.png" width="750px"/>
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<h3>Reference</h3>
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<p>Mitchell R. O’Connell, Benjamin L. Oakes, Samuel H. Sternberg, Alexandra East-Seletsky, Matias Kaplan & Jennifer A. Doudna (2014). Programmable RNA recognition and cleavage by CRISPR/Cas9. Nat Commun. doi:10.1038/nature13769</p>
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<b>BBa_K2381024</b>, is proved by OD measurement after cotransformed with plasmid containing dCas9. Its effect is even better than rapamycin, a well-used chemical molecule for replication inhibition.
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<p>Alexandra E. Briner, Paul D. Donohoue, Ahmed A. Gomaa, Kurt Selle, Euan M. Slorach, Christopher H. Nye,Rachel E. Haurwitz, Chase L. Beisel, Andrew P. May, and Rodolphe Barrangou (2014). Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality. Molecular Cell 56, 333–339, October 23, 2014 ª2014 Elsevier Inc. 333</p>
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<img src="https://static.igem.org/mediawiki/parts/2/26/T--HZAU-China--gRNA_qPCR.png" width="750px"/>
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Further investigation is conducted by qPCR and flow cytometric.
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<img src="https://static.igem.org/mediawiki/parts/f/f1/T--HZAU-China--light_gRNA.png" width="750px"/>
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Its function after light-induced promoter, <b>BBa_K2381023</b>, is proved by OD measurement.
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<h3>References</h3>
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1. Wiktor, J., Lesterlin, C., Sherratt, D. J., & Dekker, C. (2016). CRISPR-mediated control of the bacterial initiation of replication. Nucleic Acids Res, 44(8), 3801-3810.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 01:00, 18 October 2018


3'-OriC gRNA

It’s a gRNA sequence containing 20bp targeting at oriC and a full-length gRNA scaffold. The sequence is central to our project and is related with all the system built this year.
The targeting sequence of this part is acquired from reference paper1.


BBa_K2381024, is proved by OD measurement after cotransformed with plasmid containing dCas9. Its effect is even better than rapamycin, a well-used chemical molecule for replication inhibition.



Further investigation is conducted by qPCR and flow cytometric.



Its function after light-induced promoter, BBa_K2381023, is proved by OD measurement.


References

1. Wiktor, J., Lesterlin, C., Sherratt, D. J., & Dekker, C. (2016). CRISPR-mediated control of the bacterial initiation of replication. Nucleic Acids Res, 44(8), 3801-3810.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 4
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]