Part:BBa_K4800012
ackA-pta sgRNA
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 Escherichia coli NT1003 genome to assist the Cas9 protein in silencing the gene.
Build:
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. 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. 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.
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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