Analysis of cellular physiology of recombinant escherichia coli cells during synthesis of foreign protein using flow cytometry.

The production of proteins in recombinant bacteria is of increasing industrial importance. One of the main problems encountered in the fermentation of recombinant bacteria is the overburdening of the cellular metabolism caused by the synthesis of the foreign protein at high expression rates. The subsequent cell death gives one only a short time of using the cellular capacity for protein synthesis. One of the few possibilities to characterize the stress during induction of foreign protein synthesis on a single cell basis, is flow cytometry. We want to establish combinations of the present methods and to evaluate their use for the routine analysis of cell populations in bioreactors. Furthermore, we would like to take advantage of the sort function of the flow cytometer to select for strain which can combine a high specific production rate (as measured by a high content in specific protein over total protein content) with continued cell growth even after induction of foreign protein synthesis. This finetuning of production rate will be achieved mainly by mutations in the translation initiation region.