Dear Zhaowei! I have some experience of dealing with Bio-Rad electroporation instrument. I transfected bacteria and mammalian cells. Maybe you'll find my advice useful.

You should keep in mind that electroporation can seriously damage cells. So, first of all, check viability of your cells after the electroporation procedure. It seems to me your regimen is too harsh, so the amount of living cells is too small.

Also cells must have enough time to express the gene of antibiotic resistance before being selected. It takes 30 min in E.coli cells and 24-48 hours in mammalian cells. I don't know exactly how much time is needed to Pichia, but 1 hour may be too little.

Good luck!

Try centromeric vector, pPICHOLI, if you do not need stable integrants.Random integration does not work very well.

Dear Zhaowei! I have some experience of dealing with Bio-Rad electroporation instrument. I transfected bacteria and mammalian cells. Maybe you'll find my advice useful.

You should keep in mind that electroporation can seriously damage cells. So, first of all, check viability of your cells after the electroporation procedure. It seems to me your regimen is too harsh, so the amount of living cells is too small.

Also cells must have enough time to express the gene of antibiotic resistance before being selected. It takes 30 min in E.coli cells and 24-48 hours in mammalian cells. I don't know exactly how much time is needed to Pichia, but 1 hour may be too little.

Good luck!

It's hard to say from here, it all looks fine. Things you may try:

- Take the time to calibrate your spectrometer so that you use the right culture density to prepare the cells (In my experience this can make quite an impact)

- Increase the amount of DNA (I've used up to 20 ug). Also, check that the DNA precipitation actually worked.

- The voltage seems a tad high. Most manufacturers recommend 1500 V for 0.2 cm cuvettes.

- Try the LiAc/DTT method of Wu and Letchworth (PMID 14740498). It works very well.

- Just in case, increase the recovery period to 2-3 h.

- Set up a non-selection plate (without antibiotics or with His, depending on the case) as a control.

Hope it helps

Hi,

I have three suggestions:

1. Increase the amount of DNA and it will be much better if you column purify the DNA. DNA precipitation followed by ethanol wash might contains salts which will decrease the Electroporation efficiency.

2. Increase the recovery time after the electro pulse. In case of S.cerevisiae and C. albicans the recovery period is 4 hours.

3. What is the OD at 600 when you harvest the cells from secondary culture ? Use cells which have OD of 1.6 to 1.8. after extensive washing the cells number at final stages might be low so begin with higher OD of cells.

It is a while ago that I worked with Pichia, but as already mentioned before:

- increase the amount of DNA: the Invitrogen protocol recommends 5 -10 µg!!

Low DNA amount was my problem as well. In the end I also purified the DNA via appropriate columns and increase the recovery time after electroporation.

(Make sure that you keep the plates at 30°C)

Parameters of your electroporation look fine and demonstrate that you have good cell suspension and clean DNA solution. 2000 V is a little bit higher than it requires for a 2 mm cuvette but until you have not observed an arch, it is acceptable. Perhaps slightly lower electric field, 1500 V, could be better but I do not think this is a crucial factor in your experiments. Normally good E-comp Pichia cells require much less than 1 ug of plasmid DNA and generate more than 1e4 transformants per 1 ug.

The main question is whether your comp cells are really E-competent. A control transformation with an ARS containing Pichia plasmid can help you to figure it out. The transformations of both original plasmids without insertion might help you to answer two questions: 1) are the cells are competent or not? 2) does the inserted fragment is neutral for Pichia cells or it reduces the transformation efficiency either by expression of a toxic product or by functioning as a cryptic ARS which dramatically decrease the frequency of recombination.

I agree with Prashant Desai that the cell OD can be an important factor however his estimation is not quite correct for Pichia: the final OD600 of a Pichia culture before harvesting must be 1, maximum 1.5. The competency of overgrown cells is reduced dramatically. And the recovery time as Prashant mentioned must be longer - between 3 and 4 hrs with shaking at 100 RPM. You did not mentioned what you have plated but I'd recommend to plate the entire sample. You can divide it in proportion 1 and 9 for two plates.

By the way, the Invitrogen's is not quite good so you better look for some other literature sources describing procedures for preparation of Pichia electrocompetent cells (for instance Pichia protocols).

I meant "the Invitrogen's protocol"

I just transfected P. pastoris and was very successful. In addition to the suggestions giving above, i suggest that you take critical look at the following steps:

1. Preparation of the competent cells ( plate your competent cell without transfection on media with antimicrobial agent to make sure you have a competent cell)

2. Transfecting Pichia require very high con centration of DNA (5-15ug) to be successful

3. Make sure the vector is duely linearized, that is Sac1 is working well and your vector linear.

4. 1500V is usually the best for yeast electroporation

5. Be sure to use appropriate concentration of the antimicribial agent for the selective growth and dont forget to grow it 30C.

All the best.

(kind answer of my colleague Lionel, translated)

For Pichia, it's ok to follow strictly the protocol from Invitrogen EasySelect Pichia Expression Manual, woth the protocol of electroporation (position standard Sc2 of an electroporator = Saccharomyces cerevisiae 2 mm).

My colleague is using 10 µg DNA (circular), because he looks for transformants called "jackpot" with "multiple insertion on AOX1 gene". Selection with Zeocin 500 µg/ml on plates YPDS (Sorbitol).

This protocol require addition of 1ml Sorbitol 1M after transformation. It seems that it is rather important to wait one or two hours before plating after transformation!

Colonies become visible after 3-4 days on Zeocin 500. Around 10 colonies per dish with a high frequency of multiple transformants (expression of the candidate gene until 10x compared to a selection on Zeocin 100).

Good luck, Sabine (just a translation of the experience of my colleague Lionel)

Evidently something went wrong in the digestion (normally digestion did not take place to the required extent)

Hi,

before testing a lot of different conditions, add YPD/1M Sorbitol and let shake for additional 4h after elctroporation (as manual says). It may take 4 to 10 (!) days until you see colonies. So 4 days is the earliest...

And there are many others reasons why it doesn't work as discussed before...

(by the way: pPICZ and a His-mutant (GS115) may not be optimal, don't forget to add Histidin to the plates as it isn't provided by the vector or strain, if it's not a double-transformation ;-)

Good luck!

You can try to transform using LiCl method, and/or to give more incubation time

For Pichia I would suggest to prepare electrocompetent cells as described here:

http://www.biotechniques.com/multimedia/archive/00003/BTN_A_05381BM04_O_3873a.pdf (Table 1, harvest cells at an OD600 of 1.5 - 2.0).

Personally, I usually use 2.5 - 5 µg of linearized DNA and the cfu count after transformation is high (>80 when plating 10 % of the cell suspension), even with this rather low amount of DNA. Regarding recovery (YPD or 1 M Sorbitol), I would also suggest to incubate the cells at least for 2 hours before plating them on selection medium.

Do you also have problems to get positive transformants when using linearized empy vector plasmid?

Dear Zhaowei Wu,

Try to increase the vector amount!

It's also good to check for possible toxicity of your gene expression in P pastoris.

In 3 days after transformation you should start to see the colonies

Bests.

I would not worry about the toxicity of the gene driven by AOX1 promoter which presents in both vectors unless one uses methanol for induction. The promoter is supposed to be repressed very strongly under the transformation conditions.

Dear Wu,

We do the transformation of pichia pastoris more often i dont find any problem. I ask you to follow some suggestions to you.

1) Use empty plasmid as control for the transformation. I know that closed circular plasmid is equally effcient in the transformation of Pichia similar to linearized plasmid.

2) OD600 should not exceeed 1. If possible again make main culture 2 ending cells OD600=1

3) electrocompetent cells are very sensetive use ice cold sorbitol and water for washing the cells.

4) Regenrate the cells in 1M YPS with a moderate shaking

5) plate the regenrated competent cells only over YPD plate to test the viability of your competent cells

Hi Zhaowei

don't know if this problem is stil urgent for you...

The invitrogen protocol usually works, and the sorbitol transformstion is a quite propper and straight process. The selection is strict, so you don't get as much clones like for a usual Coli-Transformation. The critical step for the electroporation is the remaing salt, too much can damage your cells. If the sac1 linearization fails, you will not get a homogenous recombination....you might check the linearisation with electrophoresis, the uncut plasmid should move better than the linearized one. You can double the growth time after electroporation to two hours or wait for more time after plating, 1-2 weeks can be possible. Try reduced Zeocin-plates too, maybe the conditions are too strict...like half of the proposed concentration, the Zeocin is the most expensive part of the kit and you can plate more this way.