Can old premixed lithium acetate buffers be used for preparing and transforming Saccharomyces cerevisiae?

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Stock buffers of TE, Lithium Acetate, and PEG can be stored. However the combined solution which is used to prepare the cells for transformation must be made fresh every time. There is a loss in transformation efficiency if the solutions are not freshly prepared.

Answer Id: 3732

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My spheroplasting of Pichia worked twice, but hasn't worked since. The OD of the culture simply does not drop.

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Here are some things to consider:
(1) If the OD of cells that are used is too high, they will not spheroplast. Do not overgrow cells.
(2) Do not use old cells and make sure that they are in log phase of growth.
(3) Make sure to mix zymolase well before using. Zymolase is more of a suspension than a solution.
(4) Make the PEG solution fresh each time and check the pH.

Answer Id: 3737

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3db11d259a9db7fb8965bdf25ec850b9_FAQ

What causes the media to turn green during Pichia fermentation?

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The green color found in the supernatant of methanol grown, high cell density Pichia fermentations is due to alcohol oxidase (Aox1p). Aox1p assembles into a homo-octamer and binds flavin adenine dinucleotide as a prosthetic group in vivo. At high concentrations, the enzyme can form a crystalloid to produce a chromophor that is green. Up to 30% of total cell protein is Aox1p, when Pichia cells are grown on methanol. Aox1p, while not a secreted enzyme, will accumulate in the culture supernatant due to leakage from the cells or lysis.

Pichia can be grown to the same high densities on other carbon sources such as glycerol or dextrose without developing this color. The alcohol oxidase production is either uninduced or tightly repressed under these conditions.

To successfully purify a protein away from Aox1p (and FAD), you can use either a S column (reverse cation exchange column), or a Q column (Sepharose Fast Flow ion exchange column). The Q column is not always as efficient as an S column unless it is a large diameter and long column. Purification of His-tagged proteins on ProBond would also effectively remove the Aox1p.

Answer Id: 3739

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20885c72ca35d75619d6a378edea9f76_FAQ

How can I convert OD units to approximate Pichia cells/ml (or density)?

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An OD600=1 is equivalent to 5 x 10E7 Pichia cells/ml. After overnight (O/N) growth from a colony pick, a Pichia culture generally reaches OD 1.3-1.5.

Answer Id: 3748

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Does Pichia secrete proteases in the media? What protease inhibitors can be used to prevent proteolysis?

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Yeasts in general are known to secrete proteases. There are some proteins specifically susceptible to proteases that have optimal activity at neutral pH. If this is the case, expression using unbuffered media may be indicated. As Pichia expression progresses in an unbuffered medium such as MMH (minimal methanol plus histidine), the pH drops to 3 or below, inactivating many neutral pH proteases. Although the acidic environment of the culture should prevent activity of neutral proteases, you may use PMSF and EDTA at a 1mM concentration in Pichia crude supernatant (refresh the PMSF every few hours) and then monitor for protease activity. See Deutscher(1990) Guide to Protein Purification, Methods in Enzymology for details.

In contrast, it has been reported that by including 1% Casamino acids (Difco) and buffering the medium at pH 6.0, extracellular proteases were inhibited, increasing the yield of mouse epidermal growth factor. Please see Clare, J. J. et al. (1991) Gene 105:205-212.

Additionally, major vacuolar proteases may be a factor in degradation, particularly in fermentor cultures that have the combination of the high cell density and lysis of a small percentage of cells. Using a host strain that is defective in these proteases may help reduce degradation. SMD1168 and SMD1168H are protease-deficient Pichia strains that are defective for Pep4p, a proteinase that is required for the activation of other vacuolar proteases, such as carboxypeptidase Y and proteinase B. Please see Higgins D. R. and Cregg, J. M. (1998) Pichia Protocols. Humana Press, Totowa, New Jersey. Please note that SMD1168, SMD1168H and the Pichia Protocols book can be ordered from Invitrogen™.

Answer Id: 3749

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f9beb1e831faf6aaec2a5cecaf1af293_FAQ

What anti-foam can you use with Pichia fermentation?

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Most researchers prefer MAZU DF 204 or KFO 673. Antifoam 289 may be used, though it will not last very long. Use the minimum amount of antifoam to control foaming. A healthy five liter fermentation will require from 0 to a few milliliters of antifoam. Preferably add via a well regulated antifoam controller. If the culture foams excessively it is a sign of carbon source limitation, low pH, or ill health of the culture.

Answer Id: 3750

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685ac8cadc1be5ac98da9556bc1c8d9e_FAQ

What is the Nitrogen source in the media for fermentation?

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During the Pichia fermentation process, the nitrogen source is the ammonium hydroxide used to adjust the pH. There is no nitrogen in the basal or trace salts.

Answer Id: 3751

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21ce689121e39821d07d04faab328370_FAQ

What pattern of oxygen uptake should I expect to observe during a fermentation run?

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

For a Mut+ you should expect that initially (in the first 2-4 hours of induction) the oxygen uptake rate of the culture would be lower than the end of 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 one can expect that the oxygen uptake rate will be lower than the end of glycerol batch phase through out most of the fermentation. One has to be very careful not to poison MutS clones.

Answer Id: 3752

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48df7b8e8d586a55cf3e7054a4c85b30_FAQ

What can be used as an acid to adjust the pH of fermentation media? Does it even need to be pH'ed?

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No acid is required for Pichia fermentation. A healthy culture always acidifies the media. If the pH of the culture is increasing it is a sign of carbon source depletion or ill health of the culture.

Answer Id: 3753

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258e130476290221f597c56d351224b6_FAQ

What is the advantage of mixed feed in Pichia fermentation?

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

Answer Id: 3754

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Can YPD be used instead of BMGY-type media for pichia fermentation?

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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. The richer media makes it difficult to purify secreted proteins from the media. The BMGY formulation remedies both of these problems.

Answer Id: 3755

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cd3bbc2d7ca1bbdc055acf58609e6c24_FAQ

Do you need to add sulfuric acid to the Fermentation PTM Trace salts?

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

Answer Id: 3756

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91f9fec9b080c74297a55c392b5f40a4_FAQ

Is there a functional difference between using peptone or tryptone for Pichia media?

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Bacto-Tryptone and Bacto-Peptone are two different and specific types of peptones. Bacto-Tryptone is a slightly poorer nitrogen source, and more of the nitrogen is provided by tyrosine and tryptophan. When comparing the two as components of media for Pichia growth, growth curves may differ slightly, but there should be only minor differences between the two. In Pichia media formulations that include Yeast Nitrogen Base as a primary source of nitrogen, such as BMGY and BMMY, there should be very little or no difference.

Answer Id: 3758

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3df07fdae1ab273a967aaa1d355b8bb6_FAQ

Will Pichia pastoris vectors (e.g. pPICZ, pPIC6, pPIC9K, pPIC3.5K, pAO815) work in Pichia methanolica? Is the TEF1 promoter functional in Pichia methanolica?

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No, Pichia pastoris vectors will not work in Pichia methanolica, both have AOX1 promoters but they are not homologous so the plasmid won't be able to integrate or replicate in Pichia methanolica. TEF1 promoter is probably functional in Pichia methanolica.

Answer Id: 3759

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cbf22ab286e2ad4900bdf5d6a2e47009_FAQ

What concentration of casamino acids should be used to inhibit protease activity in Pichia expression experiments?

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In following reference, 1% Casamino Acids were used: Clare JJ, et al. Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene. 1991 Sep 15;105(2):205-12. PMID: 1937016; UI: 92039033.

In this paper, the researchers found that although Pichia grew to a similar cell density in both YP and YNB, only a very low level of mouse epidermal growth factor (0.07 µg/ml) was present in supernatants from single copy transformants when grown in YNB, and this decreased during further incubation. By using YNB medium which had been buffered to pH 6.0 and supplemented with 1% Casamino acids, secreted mEGF levels substantially increased to about 1.9 ug/ml for single copy transformants.

Answer Id: 3761

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