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.