3.1 Directed evolution and isolation of protein production strains
We applied repeated fed-batch-like cultivations in a 48-well microbioreactor system in order to characterize mutation patterns triggered by recombinant protein production under long-term cultivations. Twenty-four wells were inoculated with the host RNAP-dependent A1 expression system (BQ<A1>) and 24 with the T7 RNAP-dependent BL21(DE3) expression system (B3<T7>), in which both strains have the GOI integrated into the chromosome. GFP-producing cells were induced with 10 µmol IPTG/g CDM at the beginning of each passage, which corresponds to full induction of recombinant protein production. Fab-producing cells were induced after 6 hours of the first passage and cultivated in medium that already contained 10 µmol IPTG/g CDM in all other passages. Cells were passaged several times until no difference in growth behavior was observed. We performed the experiments with two different model proteins, the easy-to-produce protein GFPmut3.1 and challenging protein dFTN2. For GFP-producing strains, the fluorescence of GFP was used to distinguish between producing and non-producing clones over a period of six passages and a total of 42 generations. Fab-producing cells were cultivated over a period of three passages (21 generations) and dot blot analysis performed to detect producing clones.