Mini kit 1

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.

ITS sequences were amplified from the nine Synechococcus and Prochlorococcus strains (Table 1) using the qPCR condition described above. qPCR products were gel purified and cloned into the pMD18-T vector, and five clones for each strain were screened by PCR with the primers M13F (5′-TGTAAAACGACGGCCAGT-3′) and M13R (5′-CAGGAAACAGCTATGACC-3′). One positive clone for each strain was grown in 5 mL LB medium, and plasmid DNA was extracted using the E.Z.N.A. plasmid minikit I (Omega Bio-Tek). DNA fragments were amplified using the primers M13-F and M13-R with plasmid DNA as the template. PCR products were gel purified, and the DNA concentration was determined using Qubit 3 with the reagent from Invitrogen. The purified DNA was 10-fold serially diluted and served as qPCR standard. Each of the diluted standards with molecular concentrations from 10¹ to 10⁷ copies/μL was measured in triplicate. The standard curve was generated by using the CFX Manager software.