Detection of integration frequency catalyzed by integrase using phenotypic screening method / 中华微生物学和免疫学杂志

ABSTRACT

Objective To establish a system for detecting integration frequency of antibiotic resist-ante integron.Methods We cloned integron and aadA2 gene cassette into different sites of plasmid pACYC 184,and the plasmid was transformed into E.coli BL21(DE3)containing plasmid overexpressing integrase.The positive clone was cultured overnight and then was spread on LB agar plate with or without streptomycin respectively,and with appropriate amount of bacteria.Clones after cultured overnight were counted to detect the integration frequency.Meanwhile we used positive clones in LB agar plate containing streptomycin as templates to carry out PCR.The purified PCR products were sequenced to identify the integration sites.Re-suits The integration frequency of integron capturing aadA2 gene cassette in BL21(DE3) host was 1.1 x 10-3 mainly at attI site.Conclusion This system can be used to detect integration frequency.