title: GalliumHigh_4
organism: Pseudomonas aeruginosa PAO1
description: Protocols: To isolate random clones from evolved lineages, we streaked out an aliquot of the final day freezer stocks of the experimental evolution onto LB plates. After overnight incubation at 37C, we randomly picked five colonies from each evolved lineage (i.e. 200 clones in total). To assess the variation in growth and pyoverdine production of these evolved clones, we measured their performance under the treatment conditions they experienced during experimental evolution in 3-fold replication, in the same way we did for the population-level analysis. Moreover, we performed an in-depth phenotypic analysis for 16 randomly picked single clones (four per drug treatment) that were selected for sequencing Experimental evolution took place in 190 l iron-limited human serum distributed on the inner wells of a 96-well plate. We exposed wildtype cultures of PAO1 to experimental evolution for a period of 20 days under five different selective regimes in eight-fold replication each. We had a no-drug control treatment in addition to a low and a high concentration drug treatment for both gallium and flucytosine. The concentrations used were inferred from the dose-response curves, and were 50 M and 280 M for gallium and 75 M and 1 mM for flucytosine. We used the GenElute Bacterial Genomic DNA kit (Sigma Aldrich) for DNA isolation DNA concentrations were assayed using the Quantifluor dsDNA sample kit (Promega) and samples were sent to the Functional Genomics Center Zurich for library preparation (Nextera XT DNA Library Prep Kit)