Part:BBa_K4800013
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.
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:
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:
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
- 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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