Part:BBa_K4800013
YcjQ sgRNA
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.
Build:
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. 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. 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.
Test:
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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