a, Serial passaging of 1 μl of culture mixture into 120 ml fresh medium, every 12-16 h. Replica flasks 1 to 7, and passage rounds R0 to R55, are indicated. Clones related to glnA(glnG)-hns (green) and flhD-glnG (yellow) were selected. b, Clones surviving stationary phase at 37°C (Day 4, Day 8); rpoS-ompR is strongly-selected (red). c, Clones obtained in heat survival (50°C, 1 h) for 3 rounds (R3). d, rpoS-ompR plasmid (650-fold overexpressed transcript), heat selected over the ~570 constructs (3 rounds). e, Integrated rpoS-ompR (2-fold overexpressed transcript), heat selected over integrated Co (7 rounds). f, Integrated rpoS-ompR, selected over wild-type TOP10 cells (7 days, 37°C). g, ompR ORF in pBAD (430-fold overexpressed), is not heat selected over pBAD vector, (5 rounds). b-d, 10 replica flasks. e-g, 5 replica tubes, 5 PCRs per tube.