A small derivative of the broad-host-range plasmid RK2 which can be switched from a replicating to a non-replicating state as a response to an externally added inducer.
TrfA is the only plasmid-encoded protein required for RK2 replication. We report here the construction and characterization of an RK2-based vector in which trfA is expressed from the inducible promoter Pm. The resulting construct, pJBSD1, was found to replicate in Escherichia coli DH5a (recA(-)) only in the presence of a Pm inducer. In two tested E. coli recA(+) strains pJBSD1 could replicate in the absence of inducer, but a replication inducer-dependent phenotype was obtained in these strains by introducing a mutation known to reduce the trfA expression level. The plasmid construct could be used as a conditional suicide vector system for targeted chromosomal integration via homologous recombination. This feature may potentially be used for many types of studies in microbial molecular biology.
Karunakaran
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UNIGEN Center for Molecular Biology and Department of Biotechnology, Norwegian University of Science and Technology, 7489, Trondheim, Norway.
Endresen
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Ertesvåg
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Blatny
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Valla
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D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D013696:Temperature; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C067405:TrfA protein, E coli