An easily modifiable conjugative plasmid for studying horizontal gene transfer
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An easily modifiable conjugative plasmid for studying horizontal gene transfer. / Wang, Qinqin; Olesen, Asmus Kalckar; Maccario, Lorrie; Madsen, Jonas Stenløkke.
I: Plasmid, Bind 123-124, 102649, 2022.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - An easily modifiable conjugative plasmid for studying horizontal gene transfer
AU - Wang, Qinqin
AU - Olesen, Asmus Kalckar
AU - Maccario, Lorrie
AU - Madsen, Jonas Stenløkke
N1 - Publisher Copyright: © 2022 The Authors
PY - 2022
Y1 - 2022
N2 - 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.
AB - 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.
KW - Conjugative plasmid
KW - Genetic engineering
KW - Tn7 transposons
KW - λ red recombination system
U2 - 10.1016/j.plasmid.2022.102649
DO - 10.1016/j.plasmid.2022.102649
M3 - Journal article
C2 - 36100085
AN - SCOPUS:85138584451
VL - 123-124
JO - Plasmid
JF - Plasmid
SN - 0147-619X
M1 - 102649
ER -
ID: 322568662