Difference between revisions of "Part:BBa K4800012"
 
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<partinfo>BBa_K4800012 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 acka-pta SgRNA is derived from the acka and pta genes and can be localised to the acka-pta 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 ackA-pta sgRNA was derived from the ackA and pta genes and could 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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     <p style="font-size: 180%; font-weight: bold;">Build:
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     <p style="font-size: 150%; font-weight: bold;">Build:
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     <p style="font-size: 160%; font-weight: bold;">（build部分标题，可以没有，没有就把这行带着上下两个/p删了）
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     <p><center><img src="https://static.igem.wiki/teams/4800/wiki/parts/acka-pta-sgrna.png" width="50%" height="50%"></center></p>
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    <p><img src="https://static.igem.wiki/teams/4800/wiki/parts/acka-pta-sgrna.png" width="80%" height="80%"></p>
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     <div align="center">
 
     <div align="center">
 
         <strong>Fig1. ackA-pta sgRNA</strong>
 
         <strong>Fig1. ackA-pta sgRNA</strong>
 
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     <p>1.1Construction of the expression vector
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     <p>We designed the N20 sequence of ackA-pta 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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The N20 sequence of the acka-pta 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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1.2knockout experiment
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We first transformed the SgRNA and Donor of aca-pta 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 aca-pta gene.
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2 Characterization experiment
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knock-out strain fermentation test
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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 acka-pta 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 rmp.
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<p style="font-size: 180%; font-weight: bold;">Test:
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    <p style="font-size: 160%; font-weight: bold;">（Test部分标题，可以没有，没有就把这行带着上下两个/p删了）
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    <p><img src="（图片网址） " width="80%" height="80%"></p>
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        <strong>(图名表名） </strong>
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<p>（表征描述） </p >
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Latest revision as of 12:25, 12 October 2023


ackA-pta sgRNA

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


Document

Build:

Fig1. ackA-pta sgRNA

We designed the N20 sequence of ackA-pta 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]