Difference between revisions of "Part:BBa K4808003"

Revision as of 12:11, 12 October 2023

g-rhtA

The g-rhtA 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 rhtA 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

rhtA gene encodes a strong threonine exporter and transfers threonine out of the cell. The gene rhtA knockout resulted in the reduction of extracellular secretion of threonine so there are more amount of threonine can be kept inside the cell for the further production of a-KB. We design the pTarget plasmid that carrying specific gRNA sequence which can identity the rhtA 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-0 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 rhtA.

Figure 1: （A）the design of pEcCas、pTarget plasmid and donorDNA for gene knockout (B) verified the construction of pTarget-g-rhtA result through the sequencing testing. (C) colony PCR to respectively determine the knock-out of rhtA (D) verified the knock-out result through the sequencing testing

Sequence and Features