Pulsed-field Gel Electrophoresis of mcr-1 Gene

The Salmonella enterica serovar Braenderup H9812 (as size marker) and E. coli cells harboring the mcr-1 gene were fixed by SeaKem Gold Agarose (Lonza Group AG, United States) and subsequently lysed. The embedded DNAs were digested using Xba I or S1 nuclease (Takara Bio, China) in a 37°C water bath for 3 h. The restricted DNA fragments were separated in 0.5 × Tris-Borate-EDTA buffer (Sangon Biotech, Shanghai, China) at 14°C for 18 h using a pulsed-field electrophoresis system (CHEF Mapper, Bio-Rad, United States) with pulse times of 2.2 to 63.8 s. The gel block was stained and observed with a gel imager (Tang et al., 2019a (link); Liang et al., 2021b (link)). The mcr-1-specific probe was labeled using a DIG High Prime DNA Labeling and Detection Starter Kit (Roche, Sant Cugat del Vallés, Spain) following the manufacturer’s instructions. E. coli J53 was used as the recipient strain, and E. coli strains harboring mcr-1 were used as the donor strain in the conjugation transfer assay. Donor and recipients bacteria were grown together overnight and then inoculated onto LB plates with colistin and sodium azide.