3.1.1 Evolution of GFP-producing E. coli strains
Induction of cells producing GFP under the control of the A1 promoter, BQ<A1-GFP>, showed no reduction in cell growth (Figure S1). The cells were able to reach the calculated end biomass of 6 g/L CDM in each of the six passages. Similarly, the productivity remained unchanged and was always approximately 25 rfu/g CDM at the end of each passage (Figure S2). Flow cytometry revealed continuous homogeneous populations of all 24 measured cultivations, as depicted for clones #E1 and #E2 in Figure 1B.
Unlike cells producing GFP under the control of the strong T7 promoter (B3<T7-GFP>), induction of the GOI at the beginning of the cultivation led to a considerable reduction in cell growth. In passage 1, cells reached a final biomass of only 1.5 g/L CDM. However, all 24 T7 strains were able to reach the calculated end biomass of 6 g/L already in passage 2. This was accompanied by a strong reduction in productivity. As shown in Figure 1A, the first subpopulations of non-producers and weak producers appeared in passage 2 and continued to outgrow producing strains over the period of six passages, as shown by derivatives #F6 and #F7. However, in derivatives #B8, #E7, and #F6, we found mixed populations of non-producers, weak producers, and strong producing cells, which could be maintained over the whole period of six passages.
To separate single-cell colonies from this mixture, we streaked the cells on agar plates after passage 6 and analyzed the isolated colonies by flow cytometry. We isolated 10 homogeneous subpopulations from weak producing (subclone 1, S1) to strong producing (subclone 10, S10) cells (Figure 1C).