Open AccessA 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
Author(s) -
Karunakaran Ponniah,
Endresen Dag Terje,
Ertesvåg Helga,
Blatny Janet Martha,
Valla Svein
Publication year - 1999
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1999.tb08799.x
Subject(s) - plasmid , inducer , biology , homologous recombination , escherichia coli , genetics , mutation , gene , vector (molecular biology) , microbiology and biotechnology , recombinant dna
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.