Spheroplast Kit for Yeast - FAQs

通常，大菌落代表含pPIC6/pPIC6α整合体的转化株，而小菌落代表含pPIC6/pPIC6α非整合体的转化株。这些非整合体转化株已经转导了pPIC6/pPIC6α质粒，因此，在起始筛选过程中表现出低水平的杀稻瘟菌素抗性。在后续筛选中，这些非整合体转化株不会保持杀稻瘟菌素抗性。

当您为表达实验选择杀稻瘟菌素抗性转化株时，我们建议您从起始转化培养皿中挑选杀稻瘟菌素抗性菌落，然后在第二个含有适当浓度杀稻瘟菌素的YPD培养皿中划线。应选择可保持杀稻瘟菌素抗性的转化株用于下一步研究。

•应确保收集的是处于对数生长期的细胞（通常OD <1.0）。
•如果使用电穿孔法，应查看电穿孔仪使用手册中的推荐条件。可根据需要，改变电穿孔参数。
•使用更多的DNA。
•使用新鲜配制的感受态细胞。
•如果使用LiCl转化法，可尝试煮沸载体DNA。

应考虑以下几点：

1.如果所用细胞的OD值过高，则它们不能形成原生质球。不要使细胞过度生长。
2.不要使用衰老的细胞，应确保细胞处于对数生长期。
3.使用前，将酵母裂解酶混合均匀。酵母裂解酶更大程度上是一种悬液，而非溶液。
4.每次都使用新鲜配制的PEG溶液，并检查pH。

pGAP克隆可使用以下高细胞密度实验方案。加入碳料，直至达到所需的细胞密度（细胞湿重（WCW）为300-400 克/升）。如果发酵罐中的蛋白状态良好，可增加至300–400 克/升 WCW，与甲醇诱导型克隆相似。在发酵后48小时内，可达到该密度。我们已经使用这些方案，使组成型表达载体在甘油中进行发酵，并得到良好的结果。您可能需要对发酵基础盐培养基做以下改进：

1) 在分批发酵培养基中，用2%葡聚糖代替4%甘油。
2) 在补料生长培养基中，用40%葡聚糖代替50%甘油。
3) 以12 毫升/升/小时的速度补加40%葡聚糖（Jim Cregg已经发表使用多种碳源作为底物进行表达的数据；葡聚糖带来最高表达水平）。
4) 可在培养基中加入酵母提取物和蛋白胨，维持蛋白稳定性。

警告：如果您在使用His-酵母株，使用pGAPZ转化后，酵母株依然是His-标记的。使用基本培养基发酵时，需要在发酵罐中加入组氨酸。

不能，您不能对甲醇进行高压灭菌。有两种方法可解决该问题，一定程度上取决于生物反应器的大小和使用体积。您可将甲醇稀释到工作浓度，并用适用于乙醇的过滤器进行过滤除菌，或者您可以假设甲醇是无菌的（本应该是无菌的）并将其直接稀释到无菌水中。氢氧化铵溶液也不可以进行高压灭菌。您可以假设30%储液是无菌的（没有生物能在该溶液中存活），并使用无菌水将其稀释至工作浓度。

不建议使用抗生素，因为在Pichia发酵期间，大部分抗生素会在低pH的培养基中失活。也就是说，加入氨苄青霉素或卡那霉素等抗生素，并不会损害发酵过程，但抗生素会因为低pH条件而失活，甚至沉淀出来。为得到最佳结果，应使用良好的无菌技术。

您无需在PTM1微量盐或发酵培养基中加硫酸。这样做除了可能帮助盐溶解，不会产生任何其他作用。

可以。细胞在YPD中能够良好生长，但存在两个缺点：在营养更丰富的YPD中，很难控制发泡，并且难以从培养基中纯化分泌蛋白。BMGY配方可解决这两个问题。

使用混合补料主要是为了降低对Pichia不利的蛋白的表达水平。我们通常将混合补料用于MutS克隆。目的就是保持培养物处于生长活力旺盛的状态，从而“乐于”表达蛋白。

您无需在Pichia发酵培养基中加入任何酸。健康的培养物会使培养基酸化。如果培养物pH在升高，则表示碳源耗尽或培养物不健康。

这取决于克隆是Mut+还是MutS。

对于Mut+克隆，起始阶段（诱导的前2-4小时）的培养物耗氧速率低于甘油分批发酵阶段末期。当培养物适应了甲醇后，若培养物健康（即，未被过多的甲醇毒害），则耗氧速率将显著提高。在Mut+克隆发酵期间，应进行甲醇峰值测试。

对于MutS克隆，在发酵过程的大部分时间里，耗氧速率都将低于甘油分批发酵阶段末期。实验人员应非常小心，不要毒害MutS克隆。

我们不提供任何Pichia发酵实验方案。请参考我们网站中名为“Pichia发酵指南”的文档。

以下实验方案已被多次用于Pichia pastoris。该方案使用250 mL培养物，并最终浓缩至1 mL。

1.将Pichia酵母株接种于10 mL YPD培养基中，30°C震荡过夜生长。
2.第二天早晨，检测OD600。为了使细胞在下午达到对数生长期，应稀释细胞使OD600在下午4点或5点约为3.0。
3.当OD600达到3.0左右，将250 微升培养物接种到250 毫升YPD培养基中。目的是为了在第二天早晨获得健康的对数期细胞，OD600约为1.0。
4.如果OD600约为1.0，则将细胞置于1L的瓶中以3K rpm离心10分钟。
5.轻轻重悬于250 mL预冷的dH20。
6.转移到500 毫升离心瓶中，以3K rpm离心10分钟。重复操作。
7.重悬于20 mL预冷的1 M山梨醇中，并转移至50 mL离心管中。
8.以3K rpm离心10分钟。
9.重悬于1 mL的1M山梨醇中，置于冰上。
10.每次电穿孔使用80 µL宿主酵母株。

在YPD培养皿中加入1 M山梨醇，可稳定经电穿孔的细胞，因为这些细胞对渗透压有些敏感。

尽管PEG3350已被成功用于内部使用，但PEG 4000的酵母转化效果可能是最好的。

我们建议使用电穿孔法转化Pichia。采用电穿孔法，每微克线性化DNA可产生103-104个转化株，并且不会破坏Pichia的细胞壁。如果您没有电穿孔设备，则可使用Pichia原生质球试剂盒（货号K172001）、PEG 1000实验方案（使用手册第78页）、LiCl实验方案（使用手册手册第80页）或Pichia EasyComp转化试剂盒（货号K173001）。我们不建议使用原生质球将含有抗生素抗性标记物的质粒转化到Pichia。细胞壁损坏可导致细胞对抗生素的敏感性增强，使假定的转化株在抗生素抗性基因表达前发生死亡。相反，原生质球用可用于PichiaPink载体的转化，因为这些载体是利用营养缺陷型标记物进行筛选的。

Generally, large colonies represent transformants containing pPIC6/pPIC6α integrants, while small colonies represent transformants containing pPIC6/pPIC6α non-integrants. These non-integrants have transduced the pPIC6/pPIC6α plasmid, and therefore, exhibit a low level of blasticidin resistance in the initial selection process. Upon subsequent screening, these non-integrant transformants do not retain blasticidin resistance.

When choosing a blasticidin-resistant transformant for your expression studies, we recommend that you pick blasticidin-resistant colonies from the initial transformation plate and streak them on a second YPD plate containing the appropriate concentration of blasticidin. Select transformants that remain blasticidin-resistant for further studies.

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Here are some suggestinos:

- Make sure that you have harvested cells during log-phase growth (OD <1.0 generally).
- If electroporation is being used, see the electroporator manual for suggested conditions. Vary electroporation parameters if necessary.
- Use more DNA.
- Use freshly made competent cells.
- If the LiCl transformation method is being used, try boiling the carrier DNA.

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Here are some things to consider:

- If the OD of cells that are used is too high, they will not spheroplast. Do not overgrow cells.
- Do not use old cells and make sure that they are in log phase of growth.
- Make sure to mix zymolyase well before using. Zymolyase is more of a suspension than a solution.
- Make the PEG solution fresh each time and check the pH.

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Use the following high cell density protocol for pGAP clones. Feed carbon until the desired density is reached (300 to 400 g/L wet cell weight (WCW)). If the protein is well-behaved in the fermenter, increase to 300-400 g/L WCW as with methanol inducible clones. These densities can be reached in less than 48 hours of fermentation. We have fermented constitutive expressers on glycerol using these protocols with good results. Some modifications to the Fermentation Basal Salts Medium that you might want to make are:

1) Substitute 2% dextrose for the 4% glycerol in the batch medium.
2) Substitute 40% dextrose for the 50% glycerol in the fed-batch medium.
3) Feed the 40% dextrose at 12 mL/L/hr (Jim Cregg has published data on expression using several carbon sources as substrates; dextrose gave the highest levels of expression).
4) Yeast extract and peptone may be added to the medium for protein stability.

One warning: If you are working with His- strains, they remain His- after transformation with pGAPZ. Fermentation in minimal medium will require addition of histidine to the fermenter.

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No, you cannot autoclave methanol. There are two approaches to this, depending a bit on the size of the bioreactor and the volumes involved. You can either dilute to working concentration and filter-sterilize with a filter suitable for alcohols, or you can just assume that methanol is sterile (it should be) and dilute into sterile water. For the ammonium hydroxide solution, you should also not autoclave it. You can assume the 30% stock solution is sterile (nothing should live in this solution) and dilute into sterile water to the working concentration.

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The use of antibiotics is not recommended, because most antibiotics become inactivated at the low pH of the medium during Pichia fermentation. In other words, addition of antibiotics such as ampicillin or kanamycin won't hurt the fermentation process, but because of the low pH the antibiotics become inactivated or may even precipitate out. For best results, use good sterile techniques.

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You don't have to add sulfuric acid to your PTM1 salts or fermentation medium. It would serve no purpose, other than maybe help dissolve the salts.

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Yes. The cells will do fine in YPD, but there are two drawbacks: The foaming that occurs in the richer YPD is very difficult to control, and the richer medium makes it difficult to purify secreted proteins from the medium. The BMGY formulation remedies both of these problems.

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The use of mixed feeds is mainly due for "turning down" the level of expression for proteins that are troublesome for Pichia. We have generally used mixed feeds for MutS clones. The idea is to keep the culture in a state of more active growth, and thus "happier" to express proteins.

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You need not add any acid to Pichia fermentation media. A healthy culture always acidifies the medium. If the pH of the culture is increasing, it is a sign of carbon source depletion or ill health of the culture.

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It depends whether the clone is Mut+ or a MutS.

For a Mut+ clone, you should expect that initially (in the first 2-4 hours of induction), the oxygen uptake rate of the culture would be lower than that at the end of the glycerol batch phase. After the culture becomes adapted to methanol, the oxygen uptake rate will significantly increase, if the culture is healthy (i.e., not poisoned by too much methanol). One should run methanol spike tests during fermentation of Mut+ clones.

For a MutS clone, one can expect that the oxygen uptake rate will be lower than that at the end of the glycerol batch phase throughout most of the fermentation. One has to be very careful not to poison MutS clones.

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We do not offer any protocols for Pichia fermentation. Please refer to the document titled “Pichia Fermentation Guidelines” on our website.

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The following protocol has been used numerous times for Pichia pastoris. It uses a 250 mL culture that is eventually scaled down to 1 mL aliquots of each strain.

- Inoculate 10 mL YPD media with Pichia strain and grow O/N, shaking at 30 degrees C.
- In the morning, check the OD600. To get them in log phase by the afternoon, dilute cells to hit an OD600 of approximately 3.0 at 4 or 5 pm.
- When the OD600 reaches approximately 3.0, inoculate 250 mL of YPD with 250 µL of culture. The objective is to have healthy, log-phase cells in the morning at an OD600 of around 1.0.
- If the OD600 is ~1.0, spin the cells in a 1 L bottle at 3K rpm for 10 minutes.
- Gently resuspend in 250 mL cold dH20.
- Transfer to a 500 mL centrifuge bottle and spin at 3K for 10 min. Repeat.
- Resuspend in 20 mL cold 1 M sorbitol and transfer to a 50 mL conical tube.
- Spin at 3K rpm for 10 min.
- Resuspend in 1 mL 1M sorbitol, and keep on ice.
- Use 80 µL of host strain for each electroporation.

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Inclusion of 1 M sorbitol in YPD plates stabilizes electroporated cells, as they appear to be somewhat osmotically sensitive.

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PEG 4000 seems to work best for yeast transformations, although PEG 3350 has been used in-house with success.

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We recommend electroporation for transformation of Pichia. Electroporation yields 10e3 to 10e4 transformants per µg of linearized DNA and does not destroy the cell wall of Pichia. If you do not have access to an electroporation device, you may use the Spheroplast Kit for Yeast(Cat. No. K172001), PEG 1000 protocol (page 78 of the manual), LiCl protocol (page 80 of the manual), or the Pichia EasyComp Transformation Kit (Cat. No. K173001). We do not recommend spheroplasting for transformation of Pichia with plasmids containing an antibiotic resistance marker. Damage to the cell wall leads to increased sensitivity to the antibiotic, causing putative transformants to die before they express the antibiotic resistance gene. In contrast, spheroplasting can be used for transformation of PichiaPink vectors because these vectors are selected using auxotrophic markers.

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The best method for transforming YACs into yeast is the spheroplast method. Yeast are never transformed with YAC vectors by electroporation--the vectors are too big and get sheared on the way into the cell.