Conjugation Assay Protocol in E. coli

A standard conjugation assay in E. coli was carried out as previously described [29 (link)]. Briefly, the donor VA585-22 and recipient E. coli K12-Gm^R strains were grown overnight in LB broth supplemented with meropenem and gentamicin, respectively. Conjugation was performed in a 0.22 μm sterile cellulose ester membrane filter (Merck, Germany) deposited over an LB agar plate, pouring a 1:1 ratio (50 µL) of donor and recipient strains. The plate with the filter was incubated for 3 h at 37ºC, and then half of the membrane was mixed with 3 mL of LB broth and vortexed briefly. Then, 100 µL of this suspension were plated onto LB-Gm-Mem and incubated at 37 °C overnight to select transconjugants (E. coli K12- Gm^R-Mem^R).