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 YcjQ gene in the <i>Escherichia coli</i> NT1003 genome to assist the Cas9 protein in silencing the gene.
  
 
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===Usage and Biology===
 
===Usage and Biology===
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<html lang="en">
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    <meta charset="UTF-8">
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    <meta http-equiv="X-UA-Compatible" content="IE=edge">
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    <meta name="viewport" content="width=device-width, initial-scale=1.0">
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    <title>Document</title>
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<body>
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<p style="font-size: 150%; font-weight: bold;">Design:
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    <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.
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    </p>
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    <p style="font-size: 150%; font-weight: bold;">Build:
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    <div align="center">
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        <p><img src="https://static.igem.wiki/teams/4800/wiki/parts/bba-k4800013-1.png" width="50%" height="50%"></p>
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    </div>
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    <div align="center">
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        <strong>Fig.1 YcjQ sgRNA</strong>
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    </div>
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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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    </p>
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<p style="font-size: 180%; font-weight: bold;">Result:
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    <div align="center">
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        <p><img src="https://static.igem.wiki/teams/4800/wiki/parts/k4800013-2.png" width="50%" height="50%"></p>
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    </div>
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    <div align="center">
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        <strong>Fig.2 Comparison of production between NT1003-P2 and NT1003-P2-ΔYcjQ</strong>
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    </div>
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    <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.
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    </p>
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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
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]