3.1.2 Evolution of Fab-producing E. coli strains
Fab-producing strains were cultivated in the same way as described for the GFP-producing strains, except cells were kept induced for a total of three passages. All Fab-producing clones exhibited reduced growth rates in passage 2 but were able to recover and exhibited unrestricted growth in passage 3 (Figure 2A). Dot blot analysis revealed that none of the 24 T7 clones produced Fab anymore, whereas three A1 clones exhibited the desired properties of fast cell growth and an ability to produce the challenging protein. To ensure homogeneous populations, the three A1 clones were separated on agar plates. Four colonies were picked from each plate to inoculate further cultivation in media containing IPTG to confirm their productivity. As shown in Figure 2B and C, we were able to isolate three subclones (#B4.1, #D2.4, and #E2.1) that produced Fab without reducing cell growth, but in different quantities; clone #E2.1 produced comparable amounts of Fab as the non-mutated BQ<A1-Fab> wt strain, whereas the other two subclones exhibited reduced productivity.