Escherichia coli K-12
Protocols: The TraDIS-Xpress transposon mutant library was constructed by electroporating a customised mini-Tn5 transposon into E. coli strain BW25113, with selection of consequent transposon mutants on Miller's formulation of lysogeny agar containing kanamycin (30 ug/mL). Aliquots of the transposon library were recovered from storage at -70 degrees C and approximately 10^7 cells were inoculated into 5 mL of Miller's lysogeny broth (LB) in a screw-capped 50 mL tube. For co-cultures, an equal number of P. aeruginosa strain PA14 cells from an overnight culture in LB were added. An identical co-culture was grown in parallel, except that sterile glucose was added to the medium at 4% (w/v). Cultures consisting of only the E. coli transposon library were also grown under the same conditions, in LB with and without glucose. Each culture was grown in duplicate. All cultures were incubated for 24 h at 37 degrees C with shaking at 200 rpm. Genomic DNA was extracted using a Quick-DNA Fungal/Bacterial 96 kit (Zymo Research) according to the manufacturer's instructions. DNA was fragmented using a Nextera tagmentation kit (Illumina). The fragmented DNA was amplified by PCR using oligonucleotide primers that hybridised at the transposon ends and contained an Illumina adapter sequence at their 5′ end.