| 产品名称： | 发酵基础盐培养基 |
|---|---|
| 英文名称： | Fermentation Basal Salts Medium |
| 培养基类型： | 基础培养基 |
| 级别： | AR级 |
| 品牌名称： | ELITE-MEDIA（艾礼培养基） |
| 产品目录号： | M904-01、M904-02、M904-03、M904-04 |
| 产品规格： | 250g、500g、5L（无菌液体，500ml/瓶）、20L(无菌液体，5L/桶) |
| 产品外观： | 白色晶体粉末。 |
| 颜色与澄清度： | 无色透明溶液。 |
| 保存条件： | 密封，2-25°C保存。 |
| 注意事项： | 避免摄入、吸入、皮肤接触。 |
| 相关产品： | PTM1 Trace Salts |
产品描述：
发酵基础盐培养基(Fermentation Basal Salts Medium)是pichia酵母发酵表达培养基。发酵基础盐培养基适用于pichia Pink菌株与pPINK-HC载体、pPINK-LC载体、pPINKα-HC载体等。配方与配制方法
| 成分 | 含量/L |
| 磷酸 | 85% (26.7 ml) |
| CaSO4 | 0.93 g |
| K2SO4 | 18.2 g |
| MgSO4. 7H2O | 14.9 g |
| 氢氧化钾 | 4.13 g |
| 甘油 | 40.0 g |
配制方法：
1. 向900 ml去离子水中加入26.7 ml 85%磷酸，溶解基础盐38.16 g。
2. 加入40 g 甘油。
3. 用氢氧化铵调节pH值。
4. 用去离子水定容至1000 ml。
5. 121°C灭菌20 min。
实验方法：
Medium PreparationYou will need to prepare the appropriate amount of following solutions:
. Fermentation Basal Salts
. PTM1 Trace Salts
. ~75 ml per liter initial fermentation volume of 50% glycerol containing 12 ml PTM1 Trace Salts per liter of glycerol.
. ~740 ml per liter initial fermentation volume of 100% methanol containing 12 ml PTM1 Trace Salts per liter of methanol.
Fermenter Preparation and Glycerol Batch Phase
Inoculum Seed Flask Preparation
Remember not to put too much medium in the baffled flasks. Volume should be 10-30% of the total flask volume.
1. Baffled flasks containing a total of 5-10% of the initial fermentation volume of MGY or BMGY are inoculated with a colony from a MD or MGY plate or from a frozen glycerol stock.
2. Flasks are grown at 30°C, 250-300 rpm, 16-24 hours until OD600 = 2-6. To accurately measure OD600 > 1.0, dilute a sample of your culture 10-fold before reading.
Glycerol Batch Phase
1. Sterilize the fermenter with the Fermentation Basal Salts medium containing 4% glycerol .
2. After sterilization and cooling, set temperature to 30°C, agitation and aeration to operating conditions (usually maximum rpm and 0.1-1.0 vvm air), and adjust the pH of the Fermentation Basal Salts medium to 5.0 with 28% ammonium hydroxide (undiluted ammonium hydroxide). Add aseptically 4.35 ml PTM1 trace salts/liter of Fermentation Basal Salts medium.
3. Inoculate fermenter with approximately 5-10% initial fermentation volume from the culture generated in the inoculum shake flasks. Note that the DO will be close to 100% before the culture starts to grow. As the culture grows, it will consume oxygen, causing the DO to decrease. Be sure to keep the DO above 20% by adding oxygen as needed.
4. Grow the batch culture until the glycerol is completely consumed (18 to 24 hours). This is indicated by an increase in the DO to 100%. Note that the length of time needed to consume all the glycerol will vary with the density of the initial inoculum.
5. Sampling is performed at the end of each fermentation stage and at least twice daily. We take 10 ml samples for each time point, then take 1 ml aliquots from this 10 ml sample. Samples are analyzed for cell growth (OD600 and wet cell weight), pH, microscopic purity, and protein concentrations or activity. Freeze the cell pellets and supernatants at -80°C for later analysis. Proceed to Glycerol Fed-Batch Phase.
Yield
A cellular yield of 90 to 150 g/liter wet cells is expected for this stage. Recombinant protein will not yet be produced due to the absence of methanol.
Glycerol Fed-Batch Phase
Introduction
Once all the glycerol is consumed from the batch growth phase, a glycerol feed is initiated to increase the cell biomass under limiting conditions. When you are ready to induce with methanol, you can use DO spikes to make sure the glycerol is limited.
Glycerol Fed-Batch Phase
1. Initiate a 50% w/v glycerol feed containing 12 ml PTM1 trace salts per liter of glycerol feed. Set the feed rate to 18.15 ml/hr /liter initial fermentation volume.
2. Glycerol feeding is carried out for about four hours or longer (see below). A cellular yield of 180 to 220 g/liter wet cells should be achieved at the end of this stage while no appreciable recombinant protein is produced.
Note
The level of expressed protein depends on the cell mass generated during the glycerol fed-batch phase. The length of this feed can be varied to optimize protein yield. A range of 50 to 300 g/liter wet cells is recommended for study. A maximum level of 4% glycerol is recommended in the batch phase due to toxicity problems with higher levels of glycerol.
Important
If dissolved oxygen falls below 20%, the glycerol or methanol feed should be stopped and nothing should be done to increase oxygen rates until the dissolved oxygen spikes.
At this point, adjustments can be made to agitation, aeration, pressure or oxygen feeding.
Proteases
In the literature, it has been reported that if the pH of the fermentation medium is lowered to 3.0, neutral proteases are inhibited. If you think neutral proteases are decreasing your protein yield, change the pH control set point to 3.0 during the glycerol fed-batch phase (above) or at the beginning of the methanol induction (next page) and allow the metabolic activity of the culture to slowly lower the pH to 3.0 over 4 to 5 hours (Brierley, et al., 1994; Siegel, et al., 1990).
Alternatively, if your protein is sensitive to low pH, it has been reported that inclusion of casamino acids also decreases protease activity (Clare, et al., 1991).
Methanol Fed-Batch Phase
Introduction
All of the glycerol needs to be consumed before starting the methanol feed to fully induce the AOX1 promoter on methanol. However, it has been reported that a "mixed feed" of glycerol and methanol has been successful to express recombinant proteins (Brierley, et al., 1990; Sreekrishna, et al., 1989). It is important to introduce methanol slowly to adapt the culture to growth on methanol. If methanol is added too fast, it will kill the cells. Once the culture is adapted to methanol, it is very important to use DO spikes to analyze the state of the culture and to take time points over the course of methanol induction to optimize protein expression. Growth on methanol also generates a lot of heat, so temperature control at this stage is very important.
Mut+ Methanol Fed-Batch Phase
1. Terminate glycerol feed and initiate induction by starting a 100% methanol feed containing 12 ml PTM1 trace salts per liter of methanol. Set the feed rate to 3.6 ml/hr per liter initial fermentation volume.
2. During the first 2-3 hours, methanol will accumulate in the fermenter and the dissolved oxygen values will be erratic while the culture adapts to methanol. Eventually the DO reading will stabilize and remain constant.
3. If the DO cannot be maintained above 20%, stop the methanol feed, wait for the DO to spike and continue on with the current methanol feed rate. Increase agitation, aeration, pressure or oxygen feeding to maintain the DO above 20%.
4. When the culture is fully adapted to methanol utilization (2-4 hours), and is limited on methanol, it will have a steady DO reading and a fast DO spike time (generally under 1 minute). Maintain the lower methanol feed rate under limited conditions for at least 1 hour after adaptation before doubling the feed. The feed rate is then doubled to ~7.3 ml/hr/liter initial fermentation volume.
5 After 2 hours at the 7.3 ml/hr/liter feed rate, increase the methanol feed rate to ~10.9 ml/hr per liter initial fermentation volume. This feed rate is maintained throughout the remainder of the fermentation.
6. The entire methanol fed-batch phase lasts approximately 70 hours with a total of approximately 740 ml methanol fed per liter of initial volume. However, this may vary for different proteins.
Note: The supernatant may appear greenish. This is normal.
Yield
The cell density can increase during the methanol fed-batch phase to a final level of 350 to 450 g/liter wet cells. Remember that because most of the fermentation is carried out in a fed-batch mode, the final fermentation volume will be approximately double the initial fermentation volume.
Fermentation of MutS Pichia Strains
Since MutS cultures metabolize methanol poorly, their oxygen consumption is very low. Therefore, you cannot use DO spikes to evaluate the culture. In standard fermentations of a MutS strain, the methanol feed rate is adjusted to maintain an excess of methanol in the medium which does not exceed 0.3% (may be determined by gas chromatography). While analysis by gas chromatography will insure that nontoxic levels of methanol are maintained, we have used the empirical guidelines below to express protein in MutS strains. A gas chromatograph is useful for analyzing and optimizing growth of MutS recombinants.
MutS Methanol Fed- Batch Phase
The first two phases of the glycerol batch and fed-batch fermentations of the MutS strains are conducted as described for the Mut+ strain fermentations. The methanol induction phases of the Mut+ and MutS differ in terms of the manner and amount in which the methanol feed is added to the cultures.
1. The methanol feed containing 12 ml PTM1 trace salts per liter of methanol is initiated at 1 ml/hr/liter initial fermentation volume for the first two hours. It is then increased in 10% increments every 30 minutes to a rate of 3 ml/hr which is maintained for the duration of the fermentation.
2. The vessel is then harvested after ~100 hours on methanol. This time may be varied to optimize protein expression.
