The hok/sok locus, isolated from the multiple‐resistance plasmid R1 of Escherichia coli, is very efficient at ensuring the stable maintenance of plasmids in Gram‐negative systems by killing plasmid‐free cells as they arise. To investigate independently the influence of temperature and growth rate on the effectiveness of hok/sok, continuous fermentations have been conducted with the pUC‐based, IPTG‐induced, β‐galactosidase expression vector pTKW106. At fixed temperature (37°C), decreasing the dilution rate decreased plasmid stability, and at a fixed, low dilution rate (D = 0.15/h), decreasing the temperature resulted in an increase in plasmid stability. These trends are explained by the specific β‐galactosidase activity of each continuous fermentation: higher, specific, recombinant protein expression led to decreased plasmid stability (due to either segregational or structural instability, as determined by plasmid DNA isolation). A representative fed‐batch medium produced more β‐galactosidase on a volumetric basis than M9C in the chemostat, and addition of the hok/sok locus increased segregational stability by 8–22‐fold in continuous fermentations that lacked antibiotic selection pressure and in which β‐galactosidase was constantly expressed at 12% of total cell protein for 60 h (43–47 generations).
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