Role of smpB in E. coli Antibiotic Sensitivity via CRISPR Interference

Abstract

Multidrug-resistant bacteria are on the increase, which underscores the necessity of effective treatment. E. coli smpB gene encodes a system that helps in saving stalled ribosomes during translation stress, which could help the bacteria to survive in antibiotic stress by keeping the protein synthesis process going. The purpose of this research project was to determine whether CRISPR interference (CRISPRi) of smpB can make E. coli susceptible to antibiotics and modify cell growth under the experimental conditions. CRISPRi plasmids carrying two guide RNAs that target the smpB gene were cloned and transformed into E. coli strains LC-E75 and WM6026. Colony PCR and gel electrophoresis were used to check insertion and gave bands at the expected position of 416 bp. The sensitivity to antibiotics was then determined with disks of spectinomycin, chloramphenicol and a combination of both at low and medium concentration. The measurements of zones of inhibition were performed on 42 plates with various strains of E. coli and gRNA models. The results indicated that smpB-targeting CRISPRi was linked with higher antibiotic sensitivity in WM6026 strain compared to minimal impact in LC-E75 strain. A cell viability experiment in WM6026 indicated that CRISPRi activation did not have a significant impact on the growth without antibiotics. These results indicate that smpB can be involved in the antibiotic stress response and promote the continued research of the CRISPRi-based studies of bacterial genes.