What is the size of the Pichia genomic DNA?

Product FAQ

Answer

The Pichia genome is similar to that of other yeast, approximately 1.5 x 107 bp (similar to S. cerevisiae) and contains 4 chromosomes (similar to S. pombe). Reference: Ohi H, Okazaki N, Uno S, Miura M, Hiramatsu R (1998) Chromosomal DNA patterns and gene stability of Pichia pastoris. Yeast 14(10):895-903.

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: E9488

Was this answer helpful?

Yes
No
Thank you for your response

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?

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

An OD600 of 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 (in 2-5 mL).

Answer Id: E9490

Was this answer helpful?

Yes
No
Thank you for your response

What do I do if I see a volume loss during a pilot expression of my Pichia culture?

Product FAQ

Answer

You can supplement with 10% culture volume of a 5% methanol (in water) solution to regenerate the 0.5% methanol concentration each day.

Answer Id: E9499

Was this answer helpful?

Yes
No
Thank you for your response

What is the advantage of mixed feed in Pichia fermentation?

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

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

Answer Id: E9531

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

What is the mating genotype of Invitrogen™ Pichia strains, such as GS115 and SMD1168 and PichiaPink™ strain 1?

Product FAQ

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: E3763

Was this answer helpful?

Yes
No
Thank you for your response

How can I freeze Pichia?

Product FAQ

Answer

Store glycerol stocks frozen at -80° C in 15% glycerol. Glycerol stocks are good indefinitely (unless there are numerous freeze-thaws). When making a glycerol stock, we recommend using an overnight culture and concentrating it 2-4 fold. Spin down cells and suspend in 25-50% of the original volume with glycerol/medium. It is better to store frozen cells in fresh medium plus glycerol, rather than simply adding glycerol into the overnight culture.

Answer Id: E4227

Was this answer helpful?

Yes
No
Thank you for your response

What is the codon usage for Pichia?

Product FAQ

Answer

It is doubtful as to whether codon usage plays as great a role in general, as is commonly believed. Translation initiation is probably more of a rate-limiting step than elongation.
Use the following codon usage list to design your gene in the order of preference:

Glycine: GGT or GGA
Glutamic acid: GAG or GAA
Aspartic acid: GAC or GAT
Valine: GTT or GTC
Alanine: GCT or GCC
Arginine: AGA or CGT
Serine: TCT or TCC
Lysine: AAG
Asparagine: AAC
Methionine: ATG
Isoleucine: ATT or ATC
Threonine: ACT or ACC
Tryptophan: TGG
Cysteine: TGT
Tyrosine: TAC
Leucine: TTG or CTG
Phenylalanine: TTC
Glutamine: CAA or CAG
Histidine: CAC or CAT
Proline: CCA or CCT

Answer Id: E9492

Was this answer helpful?

Yes
No
Thank you for your response

Which method do you recommend using for transformation of Pichia?

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

Is there an electroporation protocol for Pichia cells that doesn't require starting with 500 mL of cells?

Product FAQ

Answer

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.

Answer Id: E9532

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

No, Pichia pastoris vectors will not work in Pichia methanolica; both Pichia pastoris and Pichia methanolica vectors have promoters derived from alcohol oxidase but they are not homologous, so the Pichia pastoris vectors will not be able to integrate or replicate in Pichia methanolica. The TEF1 promoter is probably functional in Pichia methanolica.

Answer Id: E9509

Was this answer helpful?

Yes
No
Thank you for your response