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

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Answer

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: E3732

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62cf6ffb116fafb6a6b22f4cfb6a4870_FAQ

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

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Answer

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: E3737

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228b24391a2082cb336cf8936a20ea1b_FAQ

What causes the media to turn green during Pichia fermentation?

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Answer

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: E3739

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849494304d4da84c3fa81cd1a69cc625_FAQ

What is the highest pH that still permits growth during pichia fermentation?

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Answer

The highest pH which still permits growth during Pichia fermentation is 8-8.5.

Answer Id: E3741

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dffc1b18336e02bc802ef6ae41751fde_FAQ

How can a pH 6-7 be maintained in a Pichia culture that is grown in a shake flask?

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Answer

Add 5% ammonium hydroxide solution to maintain the pH of a Pichia culture grown in shake flasks. It is used at a 28% concentrated form in fermentors. Ammonium hydroxide also acts as a nitrogen source for Pichia cells.

Answer Id: E3742

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54bbeab8a6d20642d02b193b4d575a61_FAQ

What is the size of the Pichia genomic DNA?

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Answer

The Pichia genome is similar to that of other yeast, approximately 1.5 X 10E7 bp (similar to S. cerevisiae) and contains 4 chromosomes (similar to S. pombe).

Yeast 1998 Jul;14(10):895-903 Chromosomal DNA patterns and gene stability of Pichia pastoris. Ohi H, Okazaki N, Uno S, Miura M, Hiramatsu R Osaka Laboratories, Yoshitomi Pharmaceutical Industries Ltd., Japan.

We have clearly resolved four chromosomal bands from four Pichia pastoris (Komagataella pastoris) strains by using contour-clamped homogeneous electric field gel electrophoresis. The size of the P. pastoris chromosomal bands ranged from 1.7 Mb to 3.5 Mb and total genome size was estimated to be 9.5 Mb to 9.8 Mb; however, chromosome-length polymorphisms existed among four strains.

Answer Id: E3743

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995be751114462adb54c5246713e69c4_FAQ

Does Pichia form ethanol as a byproduct as do other yeast?

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Answer

Under the conditions in which Pichia is fermented (aerobically) ethanol is not produced. Oxidative metabolism of methanol first produces formaldehyde which is then converted to carbon dioxide. There is an assimilatory cycle involving formaldehyde too, but no ethanol is made in this pathway either.

Answer Id: E3744

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39e93155814e0c28aeea71c95b34644e_FAQ

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

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Answer

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: E3748

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15d07c9162178e1e4af70d8ed6683b6b_FAQ

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

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Answer

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: E3749

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b3f053a0e55b8bef1782e79768770e09_FAQ

What anti-foam can you use with Pichia fermentation?

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Answer

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: E3750

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8ed150de21fbbf038f25c03f819581bd_FAQ

What is the Nitrogen source in the media for fermentation?

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Answer

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: E3751

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21dd1112b76df4acb81cc85c79f868b0_FAQ

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

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Answer

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: E3752

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568d8046902152ef484db2c8ced6c611_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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Answer

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: E3753

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b99ec756ae7937c44364873280b79387_FAQ

What is the advantage of mixed feed in Pichia fermentation?

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Answer

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: E3754

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1044ce7f7a965192f55c247cb939176a_FAQ

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

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Answer

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: E3755

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d6b4b37ebbf26325c77549eeb68beeaa_FAQ