Pichia Pastoris Culture

Pichia Pastoris yeast is one of the methanol-nutritional yeasts that can utilize methanol as its sole carbon and energy source. Like other yeasts, it exists mainly as a haploid during the asexual growth period, and when the environment is nutrient-limited, it often induces the mating of two physiologically different spliced haploid cells to fuse into a diploid.

Grow Pichia cells on agar plates containing desired antibiotics (72 hours at 30℃ without shaking).

• Day 1

2 cultivation.

2.1 Add growth medium (BMGY) to the baffled flask.

2.2 Single positive colony consumption growth center.

2.3 30℃, 225rpm Kyoto clearance.

• Day 2

3 Gene expression.

3.1 Transfer the culture to a sterile 50mL falcon tube.

3.2 Centrifuge and pour off the supernatant.

3.3 Add 10 mL of sterile BMMY to the used baffled flask. Spin and transfer to the corresponding Falcon tube.

3.4 Vortex tube.

3.5 Falcon Tube Centrifuge.

3.6 Pour off the supernatant and add 10 mL of fresh BMMY.

3.7 Vortex the solution and transfer to the original flask.

3.8 225 rpm seats.

• Day 3

4 Continue to express.

4.1 Add 50 µL of 100% methanol to the flask.

4.2 Retention at 30℃, 225 rpm.

• Day 4

5 cells were harvested.

5.1 From the next day, transfer the cells to a 50 mL falcon tube and harvest by centrifugation at 100℃. 2000 x g, 5 min.

5.2 Pour off the supernatant and resuspend the cell wall-containing pellet in 1 mL of water.

5.3 Transfer to a pre-weighed 2 mL tube.

5.4 Centrifuge, pour off water and use cells for desired experiment 10000 x g, 2 min.

• Alternatives

For each step where media is added, add only 3 mL, not 10 mL. All centrifugation steps were performed at 3.000 x g (instead of 2.000 x g) and only 20 µL of sterile 100% methanol was added for methanol induction.

Grow liquid cultures with a starting volume of 3 mL in a similar procedure.

Store harvested cells at -20℃.

During the operation, attention should be paid to the aseptic operation of experimental materials and the use of high-quality reagents and consumables to avoid affecting the experimental results.