TY - JOUR
T1 - Temperature and Growth Rate Effects on the hok/sok Killer Locus for Enhanced Plasmid Stability
AU - Wu, Kuowei
AU - Jahng, Deokjin
AU - Wood, Thomas K.
PY - 1994
Y1 - 1994
N2 - 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).
AB - 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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U2 - 10.1021/bp00030a600
DO - 10.1021/bp00030a600
M3 - Article
C2 - 7765697
AN - SCOPUS:0028535142
SN - 8756-7938
VL - 10
SP - 621
EP - 629
JO - Biotechnology progress
JF - Biotechnology progress
IS - 6
ER -