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2023-08-14 Hits(215)
After the transformants have been grown on the plate for some time, Mut+ and Muts screening is performed. Pick a monoclone, streak or dot on MM and MD medium (first on MM plate, then dot on MD plate, and change toothpicks once for one clone), and incubate at 30°C for 2 days. It has been observed that growing on MD medium and not growing or growing very little on MM medium is the Muts phenotype, and the rest are Mut+ phenotypes.
1. Select a single colony, place it in a 250 ml shaker flask containing 25 mI MGY, BMG or BMGY medium, and culture at 28-30°C/250-300 rpm to OD600 to 2-6 (16-18 h);
2. Centrifuge 1500-3000g at room temperature for 5 min, collect the bacteria, and resuspend the bacteria with MM, BMM or BMMY, so that OD600 is about 1.0;
3. Put the bacterial solution obtained in step 2 in a 1l shaker flask, seal it with double-layer gauze or cheesecloth, and place it on a shaker at 28-30°C/250-300 rpm to continue growing;
4. Add 100% methanol to the medium every 24 h to a final concentration of 0.5-1.0%;
5. Take the bacterial liquid samples according to the time point, the sampling amount is 1 ml, placed in a 1.5 ml EP tube, centrifuge at the maximum speed for 2-3 min, collect the supernatant and the bacterial body respectively, analyze the expression of the target protein and the best harvest time of the bacterial solution, the time points are generally taken: 0, 6, 12, 24, 36, 48, 60, 72, 84 and 96 h;
6. For secretion expression, separate the supernatant of the sample, intracellular expression, isolate the bacterial precipitation of the sample, and quick-freeze the sample with liquid nitrogen or dry ice, store at -80°C for later use;
7. The expression of recombinant proteins can be detected and identified by SDS-PAGE, Western Blot and activity experiments.
1. Select a single colony and place it in a 250 ml shaker flask containing 25 ml of MGY, BMG or BMGY medium, and cultivate it at 28-30°C/250-300 rpm to OD600=2-6 (16-18 h);
2. Centrifuge at room temperature 1500-3000g for 5 min, collect the bacteria and resuspend the bacteria (about 10-20 ml) with 1/5 to 1/10 of the original culture volume of MM, BMM or BMMY;
3. Place the bacterial solution obtained in step 2 in a 100 ml shaker flask, seal it with double gauze or cheesecloth, and place it on a shaker at 28-30°C/250-300 rpm to continue growing;
4. Add 100% methanol to the medium every 24h to a final concentration of 0.5-1.0%;
5. Take the bacterial liquid samples according to the time point, the sampling amount is 1 ml, placed in a 1.5 ml EP tube, centrifuge at the maximum speed for 2-3 min, collect the supernatant and the bacterial body respectively, analyze the expression of the target protein and the best harvest time of the bacterial solution, the time points are generally taken: 0, 24, 48, 72, 96 and 120 h;
6. For secretion expression, separate the supernatant of the sample: for intracellular expression, isolate the bacterial precipitation of the sample, detect the sample with liquid ammonia or dry ice after quick freezing, and store at -80°C for later use;
7. The expression of recombinant proteins can be detected and identified by SDS-PAGE, Westerm-Blot and activity experiments.
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