Cultivation of Fluorescent Bacterial Strains

Reference laboratory strains of P. aeruginosa PAO1 (ATCC 15692) and B. cenocepacia J2315 (ATCC BAA-245) were grown in Luria–Bertani (LB; tryptone 10 g/L, yeast extract 5 g/L, and sodium chloride 10 g/L) medium (Scharlab, Barcelona, Spain) and in tryptic soy broth (TSB; casein peptone 17 g/L, soy peptone 3 g/L, sodium chloride 5 g/L, dipotassium phosphate 2.5 g/L, and dextrose 2.5 g/L) medium (Scharlab, Barcelona, Spain) at 37 °C and 30 °C, respectively. P. aeruginosa PAO1::eGFP (MK171) [23 (link)] constitutively expressing a green fluorescent protein marker from the chromosome (eGFPmut3) and P. aeruginosa PAO1 chromosomally modified by the integration of a mini-Tn7-lux constitutively expressing the luciferase genes [24 (link)] were also used, supplementing the media with 50 μg/mL gentamicin as the selective pressure. B. cenocepacia was transformed with pETS248-Tc-E2Crimson plasmid (see the plasmid construction and bacterial transformation and its low copy number vector) and growth supplementing media with 80 μg/mL of tetracycline. The strains were preserved in 20% glycerol stocks at −80 °C and were freshly revived for each experiment.