Construction of pBGC, a non-mobilisable, GFP-expressing plasmid

To fluorescently label the wild-type isolates for competition assays using flow cytometry, we constructed the pBGC plasmid, a non-mobilizable version of the gfp-carrying small plasmid pBGT²⁵ (Supplementary Fig. ³, accession number {"type":"entrez-nucleotide","attrs":{"text":"MT702881","term_id":"1939520574","term_text":"MT702881"}}MT702881). The pBGT backbone was amplified, except for the region including the oriT and bla_TEM1 gene, using the pBGC Fw/Rv primers. The gfp terminator region was independently amplified using the GFP-Term Fw/Rv primers (Supplementary Table ²). PCR amplifications were made with Phusion Hot Start II DNA Polymerase at 2 U/µl (ThermoFisher Scientific, MA, USA), and PCR products were digested with DpnI to eliminate plasmid template before setting up the assembly reaction (New England BioLabs, MA, USA). Finally, pBGC was constructed by joining the amplified pBGT backbone and the gfp terminator region using the Gibson Assembly Cloning Kit (New England BioLabs, MA, USA). Resulting reaction was transformed by heat shock into NEB 5-alpha Competent E. coli (New England BioLabs, MA, USA), following manufacturer’s instructions. Transformation product was plated on LB agar with arabinose 0.1% and chloramphenicol 30 µg/ml, and incubated overnight at 37 °C. Plasmid-bearing colonies were selected by green fluorescence. The gfp gene in pBGC is under the control of the P_BAD promoter, so GFP production is generally repressed and induced by the presence of arabinose. pBGC was completely sequenced using primers described in Supplementary Table ². We confirmed that neither pOXA-48_K8, nor helper plasmid pTA-Mob⁶⁸, could mobilised pBGC by conjugation using the conjugation protocol described above, confirming that pBGC plasmid is not mobilizable. Finally, pBGC plasmid was introduced into our isolate collection by electroporation (Gene Pulser Xcell Electroporator, BioRad, CA, USA). Of note, we were not able to obtain pBGC-carrying transformants in eight of the isolates due, in part, to a pre-existing high chloramphenicol resistance phenotype (Ec13, Kpn10, Kpn11, and Kpn19–Kpn23).