Nb bsrdi

The protocol for the detection of pCON multimers was previously established in [8, 41] . Briefly, to visualize plasmid molecules from ancestral and evolved populations, plasmid DNA was extracted from 3 mL stationary culture using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific). To remove chromosomal DNA contamination, the plasmid DNA was treated with a PlasmidSafe ATPdependent DNase (Lucigen) according to the manufacturers' protocol. Afterward, to create open circle molecules of all plasmid conformation, the plasmid DNA was nicked using the nicking enzyme Nb.BsrDI (New England Biolabs) for 30 min at 65 C. The nicked plasmid molecules were introduced to an 0.7% (w/v) agarose gel that was stained with Midori green. The samples were electrophoresed for 120 min at 4.3 V/cm and the gel was visualized on a ChemiDoc imaging system (Bio-Rad). The program Image Lab (Bio-Rad) was used for visual analysis and quantification of the plasmid molecules. The intensity of the stained plasmid DNA bands was used to quantify the relative amount of plasmid multimer-types in each corresponding sample (Figure S2).