A New Way of Stabilizing Recombinant Plasmids

概要

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A new method to stabilize recombinant plasmids extremely well was exploited using Escherichia coli Tna (trpAE1 trpR tnaA) and pSC101trpI15-14 (tetracycline resistance, whole trp operon) as a model system. We mutagenized the Tna strain carrying pSC101trpI15-14 and isolated a mutant 6F484 that stably maintained the recombinant plasmid for 100 generations. From 6F484,plasmid-free cells (tetracycline sensitive) were screened for on selctive agar plates containing fusaric acid. The host strain FA14 was found to have lost the ability for acitive transport of tryptophan, in addition to the phenotype of Trp^-. Therefore, strain FA14 could not grow normally even in a complete medium. However, when the strain was transformed with the trp operon recombinant plasmid, its growth rate was almost restored to the original level. These results suggest that the recombinant plasmid is indispensable for the normal growth of host cells like FA14. Even is plasmid-free segregants appear during the cultivation, they cannot grow so rapidly and are diluted as a minority in total population. Consequently, owing to the deficiency of both the biosynthesis and uptake of tryptophan in host strain, the trp operon recombinant plasmid can be stably maintained.
公益社団法人日本生物工学会の論文
1990-02-25