Conjugation and Electroporation Experiments with E. coli

Conjugation experiments were performed using both broth- and filter-based methods, with the azide-resistant E. coli J53 as the recipient, as described previously^{34 (link)}. Equal amounts of donor and recipient cells at the exponential stage (the optical density at 600 nm reaches ~ 0.5) were mixed and incubated at 37 °C in LB broth or on the filter that was placed on an LB agar plate overnight. Subsequently, cells were resuspended and diluted in 0.9% NaCl, and potential transconjugants were selected on LB agar plates containing 150 µg/ml sodium azide and 4 µg/ml cefotaxime. Conjugation assays were repeated with different donor/recipient ratios.
Electroporation was carried out with E. coli DH5α as the recipient. Plasmids of C. aquatica SCLZS63 were extracted using the E.Z.N.A. plasmid Mini Kit I (OMEGA, Bio-Tek, USA), verified by agarose gel electrophoresis, and then transferred by electroporation (Micro-Pulser electroporator; Bio-Rad, USA) into DH5α competent cells. Transformants were selected on LB agar plates containing 4 µg/ml cefotaxime. The presence of bla_CAE-1 in the transformant was examined by PCR assays with primers bla_CAE-F/R.