Golden Gate assembly and molecular cloning methods.

Golden Gate assembly reactions were prepared as previously described (23, 24). Approximately equimolar amounts (∼40 fmol) of destination vector and insert storage vectors were mixed with 1 μL of 10× T4 ligase buffer, 0.5 μL (200 U) of T4 ligase, and 0.5 μL (10 U) of type IIS enzyme (e.g., BsaI) in a total volume of 10 μl. Reaction mixtures were incubated for 2 h at 37°C, 5 min at 50°C, and 5 min at 80°C. Chemically competent E. coli DH5α λpir was transformed with 2 μl of the assembly reactions by the Inoue method (52), and the cells were incubated for 2 h at 37°C to allow expression of resistance genes. Cells were plated on LB agar supplemented with 100 μg/ml ampicillin and X-Gal. Inserts were validated by colony PCR of white colonies or Sanger sequencing (Genome Quebec, McGill University). Plasmids with the correct series of inserts were isolated and transformed into chemically competent E. coli NEB5-alpha cells (New England BioLabs) for quantitative exposure assays.

Traditional restriction enzyme cloning was used in the construction of destination and storage vectors. For these reactions, vector and insert DNA were digested separately for 2 h at 37°C, purified by spin column, and ligated for 16 h at 16°C before transformation into E. coli. TOPO cloning of purified Phusion PCR products into the pCRBluntII-TOPO vector (Thermo Fisher Scientific) was performed according to the manufacturer’s protocol.