Kinetic study of instability of recombinant plasmid pPLc23trpAl in E. coli using two‐stage continuous culture system

Derepression of the phage λ pL 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.

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