Stability of Recombinant Plasmids Carrying the Threonine Operon in Escherichia coli

Abstract

The stability of recombinant strains of Escherichia coli carrying hybrid plasmids comprised of pBR322 and the threonine operon (thrA, thrB and thrC) of E. coli was examined. (1) Recombinant strains were classified into two types according to the direction of the an inserted DNA fragment containing the threonine operon, which was cloned into the HindIII site of pBR322. Type I had a more stable pBR322-thr hybrid plasmid, and Type II had a less stable one and easily lost it. scLsc-Threonine productivity of Type I strains was much higher than that of Type II, presumably depending on the stability of each plasmid. (2) Repeated transfer of the host cells carrying pAJ294, a pBR322-thr hybrid plasmid obtained from a Type I strain, without any selective pressure caused a serious loss of the plasmid. Culturing at high temperature or in a poor medium accelerated the loss, and it was prevented by the addition of ampicillin or tetracycline to the culture medium. (3) pAJ294 was stable under some storage conditions, stocking on an agar medium or after lyophilization. (4) pAJ294 was fairly stable in scLsc-threonine production medium at 30°C during growth of up to approximately 17 generations without any antibiotic.