Kinetic study of instability of recombinant plasmid pPLc23trpAl in E. coli using two-stage continuous culture system.
Derepression of the phage lambda p(L) promoter on recombinant plasmid pPLc 23-trpAl caused a rapid increase of plasmid free segregants in the population. In continuous culture, increased production of trpA protein follwing derepression was accompanied by a continuous deceleration of specific growth rate. In the repressed condition, plasmid loss per generation in continuous culture decreased as dilution rate increased from 0.06 to 1.08 h(-1). Over this range, the concentration of plasmid DNA within the cell decreased eightfold corresponding to a decrease in plasmid number from 74 to 32 molecules/cell. The use of a two-stage continuous culture system coupled with a temperature sensitive expression system allows a high trpA productivity from the derepressed plasmid for more than 48 h and also offers a possibility of minimizing the instability problem of high expression recombinants. Such a system also permits the critical study of the effects of fermentation and other regulatory parameters on expression under better controlled conditions than is possible in a batch culture or single-stage continous culture.
Siegel
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University of California, Davis, California 95616, USA.
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