Difference between revisions of "Part:BBa K4800013"
 
(17 intermediate revisions by 4 users not shown)
Line 3: Line 3:
 
<partinfo>BBa_K4800013 short</partinfo>
 
<partinfo>BBa_K4800013 short</partinfo>
  
−
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.
+
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 YcjQ gene in the <i>Escherichia coli</i> NT1003 genome to assist the Cas9 protein in silencing the gene.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
Line 20: Line 20:
  
 
<body>
 
<body>
−
    <p style="font-size: 180%; font-weight: bold;">Build:
+
<p style="font-size: 150%; font-weight: bold;">Design:
−
+
     <p>YcjQ was involved in the 1,5-PDO degradation. In order to increase the yield, we decided to knock out the YcjQ gene. We decided to use CRISPR/Cas9 to construct the strain <i>E. coli</i> NT1003 ΔYcjQ.
−
     <p>< img src="https://static.igem.wiki/teams/4800/wiki/parts/ycjq-sgrna.png" height="80%"></p >
+
    </p>
 +
 
 +
    <p style="font-size: 150%; font-weight: bold;">Build:
 
     <div align="center">
 
     <div align="center">
−
         <strong>Fig.1 YcjQ sgRNA plasmid</strong>
+
         <p><img src="https://static.igem.wiki/teams/4800/wiki/parts/bba-k4800013-1.png" width="50%" height="50%"></p>
 
     </div>
 
     </div>
−
    <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.
 
−
    </p >
 
−
<p style="font-size: 180%; font-weight: bold;">Test:
 
−
   
 
−
    <p>< img src="https://static.igem.wiki/teams/4800/wiki/parts/yciq-sgrna-result.png"></p >
 
 
     <div align="center">
 
     <div align="center">
−
        <strong>Fig.2 YcjQ sgRNA fermentation result</strong>(第二个strong前面加/)
+
        <strong>Fig.1 YcjQ sgRNA</strong>
 
     </div>
 
     </div>
−
     <p>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.</p >
+
     <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.
 +
    </p>
 +
 
 +
<p style="font-size: 180%; font-weight: bold;">Result:
 +
    <div align="center">
 +
        <p><img src="https://static.igem.wiki/teams/4800/wiki/parts/k4800013-2.png" width="50%" height="50%"></p>
 +
    </div>
 +
    <div align="center">
 +
        <strong>Fig.2 Comparison of production between NT1003-P2 and NT1003-P2-ΔYcjQ</strong>
 +
    </div>
 +
    <p>The yield of 1,5-PDO produced by these strains was analyzed by HPLC. In comparison to engineered strain NT1003-P2, the deletion of YcjQ gene markedly improved the final 1,5-PDO titer, 1.75-fold higher than that in the strain NT1003-P2. The result demonstrated that NT1003-P2-ΔYcjQ performed better, and we had decided to adopt this strain in the following experiments.
 +
    </p>
 +
 
 +
 
  
 
</body>
 
</body>

Latest revision as of 12:29, 12 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 YcjQ gene in the Escherichia coli NT1003 genome to assist the Cas9 protein in silencing the gene.


Document

Design:

YcjQ was involved in the 1,5-PDO degradation. In order to increase the yield, we decided to knock out the YcjQ gene. We decided to use CRISPR/Cas9 to construct the strain E. coli NT1003 ΔYcjQ.

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.

Result:

Fig.2 Comparison of production between NT1003-P2 and NT1003-P2-ΔYcjQ

The yield of 1,5-PDO produced by these strains was analyzed by HPLC. In comparison to engineered strain NT1003-P2, the deletion of YcjQ gene markedly improved the final 1,5-PDO titer, 1.75-fold higher than that in the strain NT1003-P2. The result demonstrated that NT1003-P2-ΔYcjQ performed better, and we had decided to adopt this strain in the following experiments.

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]