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A11155

Product FAQ

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

Answer

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.

Answer Id: E9560

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Product FAQ

What is the advantage of mixed feed in Pichia fermentation?

Answer

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.

Answer Id: E9540

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Product FAQ

If there are no Zeocin™ antibiotic-containing YPD plates readily available, would it be possible to spread Zeocin™ antibiotic on top of YPD plates and still retain efficient selection of yeast?

Answer

Zeocin™ antibiotic can be spread on top of YPD plates for selection of yeast if necessary. There is a report that this works well when done with 10-15 3 mm glass beads. However, it is recommended that some optimization be performed, since top-spreading may dilute the antibiotic’s effectiveness.

Answer Id: E9497

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Product FAQ

What is the mating genotype of your Pichia strains?

Answer

All of our Pichia strains are homothallic strains. This means that they actually switch mating type with each generation. In Saccharomyces strains, this would lead to the culture rapidly becoming entirely diploid. In contrast, Pichia pastoris strains mate inefficiently to form diploids. Therefore, at any given time, the cells in the population are both “a” and “alpha” mating types.

Answer Id: E9522

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Product FAQ

Is it critical that one uses PEG 4000 for yeast transformations?

Answer

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

Answer Id: E9530

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Product FAQ

Can antibiotics be used during Pichia fermentation?

Answer

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.

Answer Id: E9543

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Product FAQ

My transformation is not working. Do you have any suggestions?

Answer

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.

Answer Id: E9561

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Product FAQ

Can I use YPD instead of BMGY-type media for Pichia fermentation?

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

Answer Id: E9541

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Product FAQ

What are the advantages of the PichiaPink™ Yeast Expression System over the EasySelect™ Yeast Expression system?

Answer

PichiaPink™ Yeast Expression System offers significant advantages compared to the original EasySelect™ Pichia system. Please see the advantages below:

- Both high and low copy enables optimization of toxic protein expression
- 8 secretion signal leader sequences
- 4 strains
- 3 protease-deficienct host strains
- Relies on adenine selection instead of an antibiotic resistance marker

Answer Id: E9481

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Product FAQ

Which method do you recommend using for transformation of Pichia?

Answer

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 Pichia Spheroplast Kit (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.

Answer Id: E9526

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Product FAQ

What is the doubling time of Pichia? How long should I wait to see colonies on agar?

Answer

Pichia has a doubling time of about 2-3.5 hours in SC media with glucose. The yeast grow slowly at 30 degrees C and it takes at least 3 days for colonies. In practice, it takes anywhere from 3 to 7 days to get nice-sized colonies.

Answer Id: E9489

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Product FAQ

Will a recombinant protein be glycosylated as it goes through the secretory pathway in a yeast cell?

Answer

A secreted protein will be exposed to the glycosylation machinery and might be glycosylated if the protein contains the standard N-linked or O-linked glycosylation amino acid consensus sequence.

Answer Id: E9502

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Product FAQ

What is the purpose of including sorbitol in the YPD plates used for plating Pichia cells after electroporation?

Answer

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

Answer Id: E9531

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Product FAQ

Can the methanol and ammonium hydroxide solutions used to prepare Pichia fermentation medium be autoclaved?

Answer

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.

Answer Id: E9544

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Product FAQ

When selecting for blasticidin-resistant transformants in the X-33 strain using pPIC6/pPIC6alpha vectors, why do I get large and small colonies on YPD plates containing 300 μg/ml blasticidin?

Answer

Generally, large colonies represent transformants containing pPIC6/pPIC6alpha integrants, while small colonies represent transformants containing pPIC6/pPIC6alpha non-integrants. These non-integrants have transduced the pPIC6/pPIC6alpha 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.

Answer Id: E9562

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