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.