Abstract
To construct wild-type E. coli irp2 gene deletion strains, CRISPR/Cas9 gene editing technology was used, and the difficulty and key points of gene editing of wild-type strains were analyzed. Based on the resistance of the CRISPR/Cas9 system expression vector, 4 strains of 41 E. coli strains isolated from Saba pigs were selected as the target strains for the deletion of the irp2 gene, which were sensitive to both ampicillin and kanamycin. Then, CRISPR/Cas9 technology was combined with homologous recombination technology to construct recombinant vectors containing Cas9, sgRNA and donor sequences to knock out the irp2 gene. Finally, the absence of the irp2 gene in E. coli was further verified by iron uptake assays, iron carrier production assays and growth curve measurements. The results showed that three of the selected strains showed single base mutations and deletions (Δirp2-1, Δirp2-2 and Δirp2-3). The deletion of the irp2 gene reduced the ability of E. coli to take up iron ions and produce iron carriers, but not affect the growth characteristics of E. coli. It is shown that the CRISPR/Cas9 knock-out system constructed in this study can successfully knock out the irp2 gene of the wild-type E. coli. Our results providing new insights into genome editing in wild-type strains, which enable further functional studies of the irp2 gene in wild-type E. coli.
Highlights
Saba pig is an excellent local breeding pig in Yunnan province in China, with wide distribution and large quantity, and is listed as a national livestock and poultry genetic resource protection breed (Lian et al 2006)
Screening of target strains and identification of irp2 gene 41 strains of wild-type E. coli were inoculated in LB medium containing ampicillin (Ampicillin concentration was 60 μg/ml), and 10 strains were sensitive to ampicillin. 10 strains of ampicillin sensitive E. coli were inoculated in LB medium, four strains of them were sensitive to ampicillin and kanamycin
The irp2 gene was found in four strains of E. coli screened by PCR amplification, which could be used as irp2 gene knock-out target strains
Summary
Saba pig is an excellent local breeding pig in Yunnan province in China, with wide distribution and large quantity, and is listed as a national livestock and poultry genetic resource protection breed (Lian et al 2006). Due to inadequate feeding management and disease prevention and control, diarrhea and death caused by pathogenic E. coli infection in Saba pigs has become an important factor seriously threatening the development of characteristic pig industry in Yunnan province. The pathogenicity and virulence of E. coli are related to the high pathogenicity island (HPI), and the pathogenicity and virulence of E. coli are enhanced due to its existence (Rakin et al 2013). HPI has a 30.5 kb functional core regions and carries irp1-irp genes related to iron element syntheses, regulation and uptake. Irp as a marker gene of HPI is an iron-regulated gene, which is only expressed in pathogenic
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
