EVALUATION OF THE HOK SOK KILLER LOCUS FOR ENHANCED PLASMID STABILITY/

The effectiveness of the hok/sok plasmid stability locus and mechanism of cloned-gene loss was evaluated in shake-flask cultures. Addition o f the hok/sok locus dramatically increased apparent plasmid segregatio nal stability relative to the hok/sok(-) control. In terms of the numb er of generations before 10% of the population became plasmid-free, se gregational stability was increased by 11- to 20-fold in different med ia in the absence of induction of the cloned-gene (hok/sok(+) plasmid stable for over 200 generations in all media tested). With constant ex pression of beta-galactosidase in the absence of antibiotic, the segre gational stability of the plasmid containing hok/sok was increased mor e than 17- to 30-fold when beta-galactosidase was expressed at 7-15 wt % of total cell protein. Although the hok/sok system stabilized the p lasmid well in four different media (Luria-Bertani (LB), LB glucose, M 9C Trp, and a representative fed-batch medium), the ability of hok/sok to maintain the plasmid with induction of the cloned gene decreased a s the complexity of the media increased. This result is better interpr eted in terms of the influence of cloned-gene expression on plasmid ma intenance; plasmid segregational stability decreased linearly as speci fic beta-galactosidase activity increased. (C) 1994 John Wiley and Son s, Inc.