Method for maintaining culture of pichia pastoris transformants by combined using dual antibiotics

[0028] Prepare 10 mg / mL ampicillin solution and 100 mg / mL geneticin solution with deionized water, and filter bacteria with 0.22 μm sterile filter membrane. Dissolve 2.5g of YEPD solid medium and 1g of agar in 50mL of deionized water, and sterilize at 115°C for 20min. After the culture medium is cooled to 50°C~60°C, add 500μL 10mg / mL ampicillin solution and 500μL 100mg / mL geneticin solution, shake well and pour the plate, so that the final working concentration of ampicillin is 100μg / mL, geneticin The final working concentration was 1000 μg / mL. Spread 50 μL of yeast solution evenly on the plate, and culture it upside down at 28°C. After 24 hours, small single colonies appeared on the surface of the medium; after 48 hours, the volume of single colonies on the surface of the medium did not change significantly, and no new single colonies appeared, which proved that the activity of Pichia non-transformants within 24h~48h was significantly affected. inhibition.

[0030] Prepare 10 mg / mL ampicillin solution and 100 mg / mL geneticin solution with deionized water, and filter bacteria with 0.22 μm sterile filter membrane. Dissolve 2.5g of YEPD solid medium in 50mL of deionized water and sterilize at 115°C for 20min. After the culture medium was cooled to 50°C~60°C, add 500 μL 10 mg / mL ampicillin solution and 500 μL 100 mg / mL geneticin solution and shake well, so that the final working concentration of ampicillin was 100 μg / mL, and the final concentration of geneticin was 100 μg / mL. The working concentration is 1000 μg / mL. Inoculate 50μL of Pichia pastoris and culture at 28°C, 200rpm. After 24 hours of liquid culture, the relative biomass of Pichia non-transformants decreased significantly, which was about 20% of that of the control group (Example 11); after 48 hours, the relative biomass of Pichia non-transformants fluctuated slightly but remained low Level, about 30% of the control group (embodiment 11).

[0032] Prepare 10 mg / mL ampicillin solution and 100 mg / mL geneticin solution with deionized water, and filter bacteria with 0.22 μm sterile filter membrane. Dissolve 2.5g of YEPD solid medium in 50mL of deionized water and sterilize at 115°C for 20min. After the culture medium is cooled to 50°C~60°C, add 500 μL 10 mg / mL ampicillin solution and 450 μL 100 mg / mL geneticin solution and shake well, so that the final working concentration of ampicillin is 100 μg / mL, and the final concentration of geneticin is 100 μg / mL. The working concentration is 900 μg / mL. Inoculate 50μL of Pichia pastoris and culture at 28°C, 200rpm. After 24 hours of liquid culture, the relative biomass of Pichia non-transformants decreased significantly, which was about 30% of that of the control group (Example 11); after 48 hours, the relative biomass of Pichia non-transformants remained at a low level, about 30% of the control group (Example 11).