Difference between revisions of "Part:BBa K4800013"
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<partinfo>BBa_K4800013 short</partinfo>
 
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SgRNA, or small guide RNA, is an important component of the CRISPR knockdown system. The N20 sequence of the ycjQ SgRNA is derived from the ycjQ gene and can be localised to the ycjQ gene in the KA30 genome to assist the Cas9 protein in silencing the gene.
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SgRNA, which is small guide RNA, is an important component of the CRISPR knockdown system. The N20 sequence of the YcjQ SgRNA is derived from the YcjQ gene and can be localised to the acka-pta gene in the <i>Escherichia coli</i> NT1003 genome to assist the Cas9 protein in silencing the gene.
  
 
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         <strong>Fig.1 YcjQ sgRNA</strong>
 
         <strong>Fig.1 YcjQ sgRNA</strong>
 
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     <p>The N20 sequence of the YcjQ gene was found on the CHOPCHOP website, designed on primers and the corresponding SgRNA was constructed by IPCR from the original SgRNA plasmid.
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     <p>We designed the N20 sequence of YcjQ by using the CHOPCHOP website (http://chopchop.cbu.uib.no), synthesized them on primers and ligated the N20 into the pTarget-sgRNA plasmid by means of PCR.
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We first transformed the SgRNA and Donor of ycjQ into the original strain of KA30 and, after verifying the success of the knockdown, we discarded the two plasmids, SgRNA and Cas9, through several rounds of delineation. Finally, we obtained the strain of KA30 with the knockdown of the ycjQ gene.
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We made the knockout strain into competent cells, and then PRSFDuet-YciA-sfp-MmCAR-YahK and PTrc99a-davB-davA-GabT were transferred into KA30 competent cells knocked out of the ycjQ gene by electrotransfer. After verifying the success of plasmid transfer, they were plated into 5 mL of KA30 seed medium at 37°C and transferred into shake flasks after 20 h. Fermentation was carried out at 37°C and 200 rpm.
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<p style="font-size: 180%; font-weight: bold;">Test:
 
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    <p><img src="https://static.igem.wiki/teams/4800/wiki/parts/yciq-sgrna-result.png" width="80%" height="80%"></p>
 
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        <strong>Fig.2 YcjQ sgRNA fermentation result</strong>
 
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    <p>The above figure shows that the production of 1, 5-PDO in the fermentation of KA30 strain with YcjQ knockout was significantly higher than that of KA30 original strain.</p >
 
  
 
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Revision as of 05:55, 11 October 2023


YcjQ sgRNA

SgRNA, which is small guide RNA, is an important component of the CRISPR knockdown system. The N20 sequence of the YcjQ SgRNA is derived from the YcjQ gene and can be localised to the acka-pta gene in the Escherichia coli NT1003 genome to assist the Cas9 protein in silencing the gene.


Document

Build:

Fig.1 YcjQ sgRNA

We designed the N20 sequence of YcjQ by using the CHOPCHOP website (http://chopchop.cbu.uib.no), synthesized them on primers and ligated the N20 into the pTarget-sgRNA plasmid by means of PCR.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]