Part:BBa_K4808005
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.
This year, our AIS-China successfully achieved the knockout of three genes on E.coli, CICC20905, a strain that has not been used in igem and has been studied less. we provide a complete set of guidelines for the use of gene knockouts for future igemers. Our contribution parts include three specific gRNA, pTargat plasmids, pEcCas plasmids needed to knock out genes. The collection is: BBa_K4808003、BBa_K4808004、BBa_K4808005、BBa_K4808009 、BBa_K4808011.
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
- 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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