Difference between revisions of "Part:BBa K4808005"

Revision as of 10:40, 11 October 2023

g-ilvB

The g-ilvB 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 ilvB gene and directs the Cas 9 protein there for gene knocking out.

Characterization

IlvBN gene encodes the acetolactate synthase that can turn a-KB into 2-acetyl-2- Hydroxybutyrate. After knocking out the ilvB 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 ilvB 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-CAS9 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 ilvB.

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

Sequence and Features