Mechanisms Of The Class 1 Integrase Excising Resistance Gene Cassettes In Bacterial

Background:Antibiotics are considered one of the greatest human discoveries of the 20th century and revolutionized modern medicine.In fact,antibiotics have become one of the most important medical interventions,but unfortunately,bacterial resistance is increasingly threatening this therapeutic achievement.The World Health Organization has listed bacterial resistance as one of the top three public health threats of the 21st century.Horizontal transfer of drug-resistant genes is an important cause of clinical drug-resistant strains.Integron is a gene element that can integrate foreign genes through site-specific recombination and make them correctly expressed.Integron has attracted attention because of its important role in horizontal transfer of bacterial drug resistance genes.Among clinical drug-resistant strains,the class 1 integron is the most widely distributed and deeply studied integron.So far,the in vitro reaction model of the class 1 integron splicing and integrated drug-resistant gene box has not been successfully established.Therefore,we conducted in-depth research on the reaction mode of the class 1 integron splicing and integrated drug-resistant gene box as well as the existence form of gene box after splicing.It provides information for building a complete integron reaction model.Objectives:1.Determine the form of free gene cassette present after integrase-mediated gene cassette shearing.2.To explore the response mode of integron shear resistance gene box:replication mode or shear mode.Methods:The attCorfF/aadA2-aadA2 gene box,plasmid replication point R6kyori,promoter Pcat,attCaadA2/3cs gene and screened marker cat gene required by PCR amplification of target fragments were used.Seamless cloning technology was used to connected the 5 fragments to pUC19 plasmid.Then,this segment was connected to the homologous arm,and the Red homologous recombination technique was used to punch the target fragment into the JM109 chromosome of Escherichia coli to construct the strain JM109I1,which was then transferred into the plasmid with high integrase expression,and the whole genome DNA and plasmid DNA were extracted,using the whole genome DNA as the template.The frequency of clipping and cyclization of gene cassettes was detected by real-time quantitative PCR,and plasmid DNA extracted was transferred into Escherichia coli expressing Π protein to observe colony growth on the plate,and the existence form of the clipping free gene cassettes was determined by realtime quantitative PCR.The constructed strain model JM 109I3 was used to transfer into the temperature-sensitive integrase plasmid with high expression,and then eliminate the temperature-sensitive integrase plasmid.The cleaved strains were screened on the streptomycin plate,and PCR and sequencing were used to determine the mode of the integrase cleaved resistance gene box.Results:1.The knock-in model strain JM 109I1 was successfully constructed,which could indicate the existence form of the cleaved free gene box.After being transferred into the plasmid with high integrase expression,the residual fragment of the cleaved aadA2 gene box was detected by real-time quantitative PCR and the cyclization of the gene box was determined.Finally,the plasmid aadA2 gene box was screened on the streptomycin plate.2.A knock-in model strain JM109I3 of integrase shear gene cassette pattern was successfully constructed,transferred into a high expression integrase plasmid,and screened on streptomycin plates for gene cassette shear occurrence strains,which were verified by PCR,and the pattern of integrase shear resistance gene cassette was:cutpaste mode.Conclusion:1.After integrase-mediated gene cassette shearing,the free gene cassette exists in the form of a loop.2.The response mode of integron shear resistance gene box was:shear mode.