pPIC9/MoL

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	The transformation was done using the electroporation procedure (Eporator Eppendorf, USA) at 1500 kV. To prepare competent P. pastoris cells, the wild type of P. pastoris GS115 was grown in YPD in a 50 mL baffle flask at 30 °C, by shaking at 250 rpm, for 24 h. Then, 500 mL of fresh YPD medium was inoculated with 0.5 mL of the 24 h culture. The culture was grown for 24 h to an OD600 = ~ 2. The cell suspension was centrifuged at 1500 × g at 4 °C for 5 min (3700, Kubota, Japan).

Protocol tips

The transformation was done using the electroporation procedure (Eporator Eppendorf, USA) at 1500 kV. To prepare competent P. pastoris cells, the wild type of P. pastoris GS115 was grown in YPD in a 50 mL baffle flask at 30 °C, by shaking at 250 rpm, for 24 h. Then, 500 mL of fresh YPD medium was inoculated with 0.5 mL of the 24 h culture. The culture was grown for 24 h to an OD600 = ~ 2. The cell suspension was centrifuged at 1500 × g at 4 °C for 5 min (3700, Kubota, Japan).

Publication protocol

The plasmid pPIC9/MoL was extracted from E. coli K12 using the QIAprep Spin Miniprep Kit (Qiagen, USA). Then, pPIC9/MoL was linearized by digestion with restriction enzyme Sal I, and incubated at 37 °C for one hour (Promega Corporation, USA). The transformation was done using the electroporation procedure (Eporator Eppendorf, USA) at 1500 kV. To prepare competent P. pastoris cells, the wild type of P. pastoris GS115 was grown in YPD in a 50 mL baffle flask at 30 °C, by shaking at 250 rpm, for 24 h. Then, 500 mL of fresh YPD medium was inoculated with 0.5 mL of the 24 h culture. The culture was grown for 24 h to an OD600 = ~ 2. The cell suspension was centrifuged at 1500 × g at 4 °C for 5 min (3700, Kubota, Japan). The cell pellet was resuspended in 500 mL ice cold sterile water. This wash step was repeated three times to remove all YPD medium from the cell pellet. Finally, the cell pellet was resuspended in 20 mL ice-cold sterile 1 M sorbitol solution. The cells were centrifuged again to obtain the pellet, and resuspended in 1 mL ice-cold sterile 1 M sorbitol solution. Subsequently, 80 μL of cells of the final cell suspension was mixed with 20 μg linearized pPIC9/MoL, and incubated at 4 °C for 5 min, and then all the contents were transferred to an ice-cold 0.2 cm electroporation cuvette. After pulsing the cells, 1 mL of ice-cold 1 M sorbitol solution was immediately added to the cuvette. Then, 200 μL aliquots of the cell suspensions were spread on RD agar plates, then, incubated for three days at 30 °C.

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