DETERMINATION OF KINETIC-PARAMETERS RELATED TO THE PLASMID INSTABILITY - FOR THE RECOMBINANT FERMENTATION UNDER REPRESSED CONDITION

Abstract

The plasmid stability under the repressed state of cloned gene was studied theoretically as well as experimentally using recombinant E. coli K12-DELTA-H1-DELTA-trp/pPLc23trpA1 as a "host-vector" model system. The important kinetic parameters studied were the plasmid loss rate (Kelvin) describing the rate at which the plasmid-harboring cells lose plasmids and the plasmid-free cells are generated per unit time and the difference in growth rates (DELTA) between the two genotypes. These parameters were carefully defined, studied, and compared with other key kinetic parameters involved in the recombinant fermentation to further our understanding of metabolism of recombinants. The ratio of the concentration of plasmid-free cells to plasmid-harboring cells (OMEGA) was introduced, and the mathematical model was derived and used for the determination of the kinetic parameters associated with plasmid instability. These methods developed based on the theoretical considerations were tested experimentally. The results of these methods were compared, and the best method was selected and recommended. The effect of temperature and dilution rate on kinetic parameters Kelvin and DELTA were also studied in continuous culture, in order to provide some practical information related to the operation and control of recombinant fermentation processes.