Kinetics analyses of plasmid instability and geneexpression in a recombinant escherichia coli using mass spectrometer

Abstract

For the kinetic analses of plasmid instability and gene expression, batch and continuous cultures, employing a recombinant Escherichia coli W3110 ΔtrpLD $trpR^{ts}$ $tna^-$/pCRT185 as a model plasmid-host cell system, were carried out. In the batch culture, the indirect estimation of cell substrate and product concentrations, which were obtained in on-line b mass spectrometric analysis of exhaust gas from fermentor provided the excellent agreements with the experimental data at both temperature of 37℃ and 42℃. From these indirect estimates various fermentation parameters were evaluated at the repressed and derepressed conditions. Under derepressed condition of 42℃ maintenance coefficient $k_1$ and yield coefficient $k_2$ were found to increase by 70-80% and 10%, respectively, than the values under repressed condition of 37℃. In addition it was also found that specific rates of oxygen uptake and carbon dioxide evolution were also increased by ca. 10% as compared with those determined from host cell culture at 42℃. Substrate consumption rate, however, showed a little difference between 37℃ and 42℃, as with maintenance energy(m) and cell yield (Y). These results suggested that the expression of foreign gene burdened the cell with extra metabolic and energy stresses. In the single-stage continuous culture at derepressed condition, 42℃, plasmid-free cells appeared more rapidly in culture broth as the dilution rate decreases. At the lowest dilution rate D=0.075 $hr^{-1}$, the productivit of cloned gene product and plasmid content showed the highest values of ca. 10 mg-trp/g-cell.hr and 0.5 mg-DNA/g-cell, respectively. Plasmid DNA content per g-cell was decreased from 0.5 mg/DNA/g-cell to .02 mg-DNA/gcell as the dilution rate increased. In order to assess quantitatively the plasmid instabilit, two parameters, i.e., the probability of plasmid loss (P) and the difference in the specific growth rate δ between the plasmid-harboring and plasmid-free cells, were estimated f...