• Microbiology and Biotechnology Letters
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• v.18 no.1
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• pp.89-93
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• 1990

To improve the stability of recombinant pBR322-trip$^+$ plasmid (pLTW24) in E. coli culture, a positive selection system was devised. A DNA fragment containing pheW$^+$ gene (a structural gene for tRNA$^{phe}$ was isolated and inserted into the pBR322-trip$^+$ plasmid(pLTP24). A temperature sensitive host strain. LC901-pheS$^{-ts}$, was constructed for this plasmid by transducing pheS$^{-ts}$ allele (phenylalanyl-tRNA synthetase) to E. coli LC901 using P1kc bacteriophage. The LC901-pheS$^{-ts}$ cells were unable to grow at a restrictive temperature when they had lost the pBR322 :: pheW$^+$ (pLTP24) plasmid. The effects of pheW$^+$ gene on the plasmid stability and the expression level of trip$^+$ gene in LC901-pheS$^{-ts}$ strain were investigated. The proportion of Trip$^+$ colonies among LC901-pheS$^{-ts}$ strain carrying plasmid pLTP24 was 99%, whereas that of LC901 strain carrying plasmid pLTW24 was 7% at the end of 20 generations. After 100 generations of growth, the strain LC901-pheS$^{-ts}$ carrying plasmid pLTP24 showed little loss of plasmids. While the majority of plasmid pLTW24 in LC901 strain were lost in the same period. The activities of tryptophan synthetase (T. Sase) and anthranilate synthetase (A. Sase) in LC901 strain carrying pLTW24 were about 1.2 times and 1.8 times respectively of those in LC901-pheS$^{-ts}$ strain carrying pLTP24.