Transformation of electrocompetent PichiaPinkTM pastoris

For transformation, a glycerol stock of strain 4 of PichiaPink^TM pastoris (Invitrogen) was streaked on an YPD-plate (1% (w/v) yeast extract (Gibco-BRL), 2% (w/v) peptone (Foremedium), 2% (w/v) dextrose, 2.4% (w/v) agar (Carl Roth), and 50 μg/ml ampicillin (Sigma-Aldrich) and grown for 3–5 days at 28°C. As a starter culture, 10 ml YPD in a 100 ml Erlenmeyer baffled flask were inoculated with a colony, cultured at 28°C and 250 rpm for 1–2 days to OD₆₀₀ 6. To generate electrocompetent cells, 100 ml YPD were inoculated to OD₆₀₀ 0.2 with the starter culture and grown for 1–2 days at 28°C and 250 rpm in a 1 l Erlenmeyer baffled flask to OD₆₀₀ 1.3–1.5. After each sedimentation step at 1,500g for 5 min at 4°C, the cells were washed twice in 250 and 50 ml of ice-cold water and thereafter resuspended in 10 and 0.3 ml of 1 M sorbitol, respectively.

For transformation 80 μl of fresh electrocompetent cells in 1 M sorbitol were mixed with 10 μg vector linearized with AflII. The cells were transferred to an ice-cold 0.2 cm electroporation cuvette and electroporated at 1,500 V using the Electroporator 2510 (Eppendorf). After pulsing, 1 ml ice-cold YPDS (YPD with 1 M sorbitol) was added, the cells were mixed and incubated for 2 hours at 28°C without shaking. Aliquots of 200 μl were spread on Pichia adenine drop-out plates (PAD: 100 mM potassium phosphate buffer (PPB), pH 6.0, 0.2% (w/v) Kaiser’s SC-Ade mixture (Foremedium), 1.34% (w/v) yeast nitrogen base (YNB, Foremedium), 2% (w/v) dextrose, 2.4% (w/v) agar, and 50 μg/ml ampicillin) and incubated at 28°C until white colonies were formed. Single colonies were re-streaked on fresh plates. Starter cultures of colonies were prepared in 10 ml Synthetic Complete Dextrose adenine drop-out medium (SCD-Ade: 100 mM PPB, pH 6.0, 0.2% (w/v) Kaiser’s SC-Ade mixture, 2% (v/v) dextrose, 1.34% (w/v) YNB, 0.0004% (w/v) biotin (Sigma-Aldrich), and 50 μg/ml ampicillin) in a 100 ml Erlenmeyer baffled flask and cultured at 28°C and routinely 250 rpm to OD₆₀₀ 6. For detection of transformed clones, 1 μl of a 1:10 starter culture dilution was used in a PCR specific for the expression cassette of pPinkα-HC (primers AOX1 and CYC1, Table 1). An aliquot of the PCR reaction was analyzed by 1.5% (w/v) Tris-borate/EDTA agarose gel electrophoresis with subsequent EtBr-staining. As a marker, the GeneRuler 1 kb DNA Ladder was used (ThermoFisher Scientific).