Enhanced plasmid stability through post-segregational killing of plasmid-free cells

In order to develop an extremely stable, inducible host/vector system, the following genetic manipulations were made: a recA mutation was introduced into the chromosome of the host strain, the plasmid selectable marker was changed from ampicillin to kanamycin, and the parB stability locus of plasmid R1 was added to the plasmid. The stability of the new vector, pTKW106, was increased such that the fraction of plasmid-bearing cells present during chemostat fermentations under selective pressure increased from 1.75% to 100% when plasmid protein production was fully induced. At this level of induction, β-galactosidase represents 10% of the total cell protein. In addition, the frequency of generation of plasmid-free cells was shown to decrease from 1.0 per generation to less than 10^-11 with full promoter induction under non-selective conditions.