A set of temperature sensitive-replication/-segregation and temperature resistant plasmid vectors with different copy numbers and in an isogenic background (chloramphenicol, kanamycin, lacZ, repA, par, polA)

• Published in: Gene 2000-01, Vol.241 (1), p.185-191
• Main Authors: Hashimoto-Gotoh, Tamotsu, Yamaguchi, Miki, Yasojima, Koji, Tsujimura, Atsushi, Wakabayashi, Yoshihiro, Watanabe, Yoshihisa
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; chloramphenicol; Chloramphenicol - pharmacology; Colony-Forming Units Assay; DNA Polymerase III - metabolism; Drug Resistance, Microbial - genetics; Gene Dosage; Genetic Vectors; kanamycin; Kanamycin - pharmacology; Kinetics; Lac Operon - genetics; lacZ gene; Mutation; par gene; plasmid pTH18cr; plasmid pTH18cs1; plasmid pTH18cs5; plasmid pTH18kr; plasmid pTH18ks1; plasmid pTH18ks5; plasmid pTH19cr; plasmid pTH19cs1; plasmid pTH19cs5; plasmid pTH19kr; plasmid pTH19ks1; plasmid pTH19ks5; Plasmids; polA gene; repA gene; Temperature; Time Factors; Transcription Factors - genetics
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/S0378-1119(99)00434-5

A set of plasmid vectors conferring chloramphenicol resistance (Cm R), 3064 bp in size, or kanamycin resistance (Km R), 2972 bp in size, were developed, having multiple cloning sites in lacZ′ genes for α-complementation. pTH18cs1, pTH19cs1, pTH18ks1 and pTH19ks1 are temperature-sensitive (ts) in DNA replication (ts-Rep); pTH18cs5, pTH19cs5, pTH18ks5 and pTH19ks5 are ts in plasmid segregation (ts-Seg); and pTH18cr, pTH19cr, pTH18kr and pTH19kr are temperature resistant (tr) in both. They are based on the pSC101 replicon consisting merely of the replication origin and repA gene, compatible with ColE1/pMB1/p15-derived plasmids, and thus do not require polA function of host cells. The copy numbers of the ts-Rep, tr and ts-Seg plasmids were 14, 5 and 1 per chromosome at 30°C, respectively. These plasmids are fairly stable when inherited at 30°C, but not above 37°C or 41.5°C, depending on the repA mutations and host strains. They are isogenic apart from the ts mutations in the repA gene, and thus provide with useful tools for having appropriate controls in various experiments including bacterial gene-targeting, transposon mutagenesis, toxic gene expression, differential substitution on host functions, gene dosage analysis and so on.