Fig. 1: Synthetic mutation assay to study evolutionary rescue dynamics in expanding microbial colonies.

a Schematic of evolutionary rescue of a resistant mutant (red) associated with a fitness cost relative to its wild-type ancestor (gray) via a compensatory mutation that renders it resistant but without a cost (blue). b Outline of experimental competition assay of resistant clones (red, 10%) interspersed in a colony of susceptible wild-type cells (gray, 90%). c Microscope images of the colony front ≈ 1 h after inoculation (day 0). Scale bar 10 μm. d Narrow but persisting resistant clone at the front just prior to application of hygromycin (day 5). Scale bar 100 μm. e Resistant growth dome after initiation of hygromycin treatment (day 6). Scale bar 100 μm. Images in c–e are representative for experiments repeated in n = 6 independent colonies. f Schematic of synthetic mutation system (see genotypes in Supplementary Table 1). A red fluorescing (RFP) and cycloheximide responsive (inactive cyh2r) genotype (top) switches to a cyan fluorescing (CFP) and cycloheximide unresponsive (active cyh2r) genotype (bottom) via loxP recombination using a β-estradiol-tunable Cre recombinase (Cre-EBD) (see Supplementary Movie 1). UBQ denotes a ubiquitin moeity that triggers proteolytic cleavage and hygMX indicates a constitutively expressed hygromycin resistance cassette. g Illustrating microscopy image of a colony one day after treatment application. Most resistant clones have been outcompeted by the wild-type (Extinction). Only one clone acquired a compensatory mutation (Rescue) and persisted to expand upon hygromycin application (Treatment failure).