Author(s)

Xuehan Wang

Corresponding Author

Xuehan Wang

Abstract

New gene associated with Pseudomonas aeruginosa aminoglycoside antibiotic resistance was discovered by applying Mu transposon recombination technology, named arcR. In this experiment, partially deleted arcR gene and its upstream and downstream fragments were amplified from the genome of Pseudomonas aeruginosa PAOI, and taking suicide plasmid pEX18Tc as vector, to construct homologous recombination vector pEX-△arcR of arcR gene, and then the recombinant vector was transferred into Pseudomonas aeruginosa PAK by means of conjugal transfer. Then, single-exchange strains were screened out through antibiotic phenotypes and genotypes PCR detection, and double-exchange strains were screened out through SacB gene sucrose lethal effect. Compared with traditional electric conversion method, this method increases the conversion rate and reduces the false positive rate, and the correct rate of PCR positive detection reaches 100%, which solves the problem that some genes of Pseudomonas aeruginosa are difficult to knock out by electric conversion method. By sequencing analysis of screened double-exchange strains, arcR gene lacks 219 bases, and ArcR gene knockout strain of Pseudomonas aeruginosa was successfully constructed. MIC (minimum inhibitory concentration) test showed that the resistance of arcR gene deleted strains to streptomycin was higher than that of wild strains.

Keywords

Pseudomonas Aeruginosa, Conjugal Transfer, Arcr, Antibiotic Resistance, Gene Knockout