Evolution of antibiotic resistance. (A) Immediately after pouring the agar layers form two distinct wedges, as shown with the dye malachite green as a visual indicator. (B) After 16 hr of incubation the two layers have intermixed to create a smooth gradient of the dye. (C) A top-down schematic, showing the site of inoculation and chemical gradient. (D) Changes in minimum inhibitory concentration following antibiotic exposure, minimum of three biological replicates. Speed-selected E. coli MG1655 cells were passed through SAGE plates containing the listed antibiotics at approximately 5x the initial MIC value. Those which evolved resistance were then passed through a separate plate containing 25x the initial MIC. Individual tests are shown in Table S1. Lineages derived from the first passage were named e.g. Poly B 1, Poly B 2, etc., and lineages built by passing evolved strains through the 25x plates were correspondingly named Poly B 1-1, Poly B 1–2, etc. *Bacteria were exposed to plates containing 10x and 50x the initial MIC of ciprofloxacin, and 20x and 100x the initial MIC of rifampicin, respectively. ‡Resistance was greater than 256x the initial MIC. Cells were uniformly resistant to 8192 mg/L of streptomycin, and poor solubility limited testing at higher drug concentrations.