Difference between revisions of "Part:BBa K4808004"

Revision as of 10:53, 12 October 2023

g-ilvI

The g-ilvI is a guide RNA that can form a complex with Cas 9 in E.coli cicc 20905. It is a specific RNA sequence (around 20 bp) that recognizes the ilvI gene and directs the Cas 9 protein there for gene knocking out.

Characterization

CRISPR-CAS 9

CRISPR-Cas 9 systems contain two components: a guide RNA and a Cas 9 protein. The gRNA is a short synthetic RNA that defines the genomic target to be modified and the Cas9 protein is a DNA endonuclease. These two components will form a complex which recognizes the PAM site and binds to the target site, and then uses the cas9 protein to cut the double-stranded DNA in the target region to break it, inducing cells to perform homologous recombination repair to achieve the purpose of gene knockout or knockin. It is one of the most scalable genome editing technologies due to the ease of generating gRNAs. Simply changing the target sequence present in the gRNA can alter the genomic target of the Cas protein thus enabling the knockout of different genes.

Our knockout method

Our achievement

ilvIH gene encodes the acetolactate synthase that can turn a-KB into 2-acetyl-2- Hydroxybutyrate. After knocking out the ilvI gene, the catabolism of a-KB can be reduced so we can accumulate more a-KB inside the cell. We design the pTarget plasmid that carrying specific gRNA sequence which can identity the ilvI gene, then we obtained donorDNA through two rounds of PCR. The donorDNA was used for homologous recombination with genomic DNA. We then put this pTarget plasmid and donor DNA into AIS-1 strains that has already carried pEcCas plasmid for the CRISPR-CAS 9 knockout experiment. (We referred to the experimental procedures published by Qi Li, Bingbing Sun, et al. in 2020) Through the results of colony PCR and gene sequencing, we confirmed the successful knockout of ilvI.

Figure 2 : （A）the design of pEcCas、pTarget plasmid and donorDNA for gene knockout (B) verified the construction of pTarget-g-ilvI result through the sequencing testing. (C) colony PCR to respectively determine the knock-out of ilvI (D) verified the knock-out result through the sequencing testing

Sequence and Features