P. pastoris GS115 was transformed with linearized pHBM306 plasmid DNA and the recombinant transformants were first selected on minimal selective MD medium.
The fermentation kinetics of ATCC 824 transformants were first investigated using a pH set-point of 5.0.
Transformants were first selected for functional β-glucosidase phenotype followed by screening for organic solvent tolerance in acetonitrile, dimethylformamide, dimethyl sulfoxide, methanol, 2-propanol.
For the measurement of bacteria growth rate, transformants were first grown at 37 °C in a shaking incubator with 3 ml liquid LB medium containing 10 g/L tryptone, 10 g/L NaCl and 5 g/L yeast extract.
The transformants were first cultivated at 30°C for 24 hours in a test tube containing 5 mL BHI medium with 25 μg mL−1 kanamycin and then inoculated into 20 mL GP2 medium (Takahashi et al. [2012]) containing 25 μg mL−1 kanamycin in a 200-mL flask for fermentation.
Transformants were first selected by the auxotrophic pDblet (uracil) marker in order to estimate transformation efficiency.
Positive transformants were first screened on THB agar plates under the selective pressure of spectinomycin.
The resulting transformants were first screened by phenotype characterization followed by PCR amplification.
Identified transformants were first surveyed for the presence of lp28-1 because this plasmid is essential for persistent infection of immunocompetent hosts [20], [29].
Transformants were first selected for Trp+ at 30°C and then toothpicked to select Ts colonies (note that the procedure cured the spectinomycin (Sp) marker of BD54 ts-6).
