An easily modifiable conjugative plasmid for studying horizontal gene transfer

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Horizontal gene transfer is an important mechanism in bacterial evolution and can occur at striking frequencies when mediated by mobile genetic elements. Conjugative plasmids are mobile genetic elements that are main drivers of horizontal transfer and a major facilitator in the spread of antibiotic resistance genes. However, conjugative plasmid models that readily can be genetically modified with the aim to study horizontal transfer are not currently available. The aim of this study was to develop a conjugative plasmid model where the insertion of gene cassettes such as reporter genes (e.g., fluorescent proteins) or antibiotic resistance genes would be efficient and convenient. Here, we introduced a single attTn7 site into the conjugative broad-host-range IncP-1 plasmid pKJK5 in a non-disruptive manner. Furthermore, a version with lower transfer rate and a non-conjugative version of pKJK5-attTn7 were also constructed. The advantage of having the attTn7 sites is that genes of interest can be introduced in a single step with very high success rate using the Tn7 transposition system. In addition, larger genetic fragments can be inserted. To illustrate the efficacy of the constructed pKJK5 plasmids, they were complemented with sfGFP (a gene encoding superfolder green fluorescent protein) in addition to seven different β-lactamase genes representing the four known classes of β-lactamases.

Antal sider10
StatusUdgivet - 2022

Bibliografisk note

Funding Information:
We thank Søren J. Sørensen for his suggestions and support throughout the manuscript. We thank Valeria Bortolaia at the Technical University of Denmark for providing strains E. coli 15083205 and E. coli 15093653 used as templates for amplification of genes bla CMY-2 and bla ampC , respectively. We thank Qiue Yang and Timothy R. Walsh team from Oxford university for providing the genes bla NDM-5 and bla OXA-181 . We thank Ana Filipa Silva for kindly providing the gene bla KPC-2 . We would like to thank the China Scholarship Council for funding QW. This work was supported by the Lundbeck Foundation ( JSM, R250-2017-1392 ) and Villum Fonden ( JSM, 00028304 ).

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© 2022 The Authors

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