Pichia EXpression

Strains

PichiaPink Strain 1 is the wild-type ade2 knockout Pichia strain. The ade2 knockoutrenders the PichiaPinkTM strain an adenine auxotroph (i.e., it requires an external adenine source for growth). These cells are unable to grow on minimal medium or adenine dropout medium, and display a slow-growth phenotype on rich medium. PichiaPink Strain 1 is the parental strain from which the rest of the PichiaPink strains are derived; therefore, all PichiaPink strains carry the ade2 deletion.

PichiaPink Strain 2 is a pep4 knockout, which prevents it from synthesizing proteinase A, a vacuolar aspartyl protease capable of self-activation. Since proteinase A also plays a role in the subsequent activation of additional vacuolar proteases, pep4 knockout strains have a diminished proteinase B activity and lack carboxypeptidase Y activity altogether.

PichiaPink Strain 3 is a prb1 knockout, which prevents it from synthesizing proteinase B, a vacuolar serine protease of the subtilisin family.

PichiaPink Strain 4 is double knock-out for both proteinases A and B (i.e., pep4 and prb1), therefore has the lowest protease activity amongst the PichiaPink strains.

Cloning (pPinkalpha-HC)

Clone gene of interest into the Xho1 and one of these sites: Stu1, KpnI, NaeI, FseI, or SwaI

For PCR cloning:
Forward primer: nnnnnn CTC GAG aaa agg ---Gene specific 20n
Rev primer use Kpn1, Fse1, Swa1 or Nae1,EcoRV with stop codon

After clone verification by PCR or digestion, use one of the following enzymes to cut plasmid

Mam I, EcoN I, Spe I, or Afl II

Alternatively, the vector can be linearized by cutting it at a unique site within the AOX1 promoter region to promote integration into the PichiaPink genome. e.g., PmeI restriction enzyme, provided that the gene of interest does not contain the PmeI recognition site

Cloning pJ912-19

Clone gene of interest into the Xho1 and one of these sites: Nhe1, Not1, Spe1

For PCR cloning:

Forward primer: nnnnnn CTC GAG aaa agg ---Gene specific 20n
Rev primer use Nhe1, Not1, Spe1 with stop codon. After clone verification by PCR or digestion, use one of the following enzymes to cut plasmid

SacI, SwaI, BamHI
*Zeocin selection in E.coli and Pichia pastoris
*AOX1 promoter for methanol inducible expression of gene of interest in Pichia pastoris
*Alpha factor signal sequence for secreted expression of gene of interest
*For integration at AOX1 locus, linearization with SwaI or SacI
+Choose SwaI to linearize vector for low to medium expression level (random integration);
+Choose SacI to linearize vector for increased expression
*For integration at AOX1 Terminator, linearization with BamHI
*To remove E. coli sequence from the integrated expression cassette, linearization with SwaI (or SacI) and PstI
*Your gene must NOT include BamHI, BsaI, PstI, SacI or SwaI sites

Purify linearizd plasmid DNA using spin column, eluted with hot water (>65 oC).

Transformation of Pichia by Electroporation (DNA2.0 Protocol)

Competent Cells

1: Grow a 5 ml overnight culture of P. or K. pastoris cells in YPD in a 30°C shaking incubator.
2: The next day, dilute the overnight culture to an A600 of 0.15 to 0.20 in a volume of 50 mL of YPD in a flask large enough to provide good aeration. (Starting volumes may be scaled up or down)
3: Grow yeast to an A600 of 0.8-1.0 in a 30°C shaking incubator, approximately 4-5 hours.
4: Centrifuge the culture at 500 x g for 5 minutes at room temperature and pour off the supernatant.
5: Resuspend the pellet in 9 ml of ice-cold BEDS solution, supplemented with 1 ml of 1M Dithiothreitol (DTT).
6: Incubate the cell suspension for 5 minutes at 100 rpm in the 30°C shaking incubator.
7: Centrifuge the culture again at 500 x g for 5 minutes at room temperature and resuspend the pellet in 1 ml of BEDS solution without DTT.
8: The cells are now ready for transformation. Alternatively, they may also be stored at -80°C  after slowly freezing them by placing the aliquots of 200 μL volume in a Styrofoam box placed in the -80°C freezer overnight. Competent cells can be stored at least 6 months at -80 oC.

Transformation

1, Mix 4 μL (50-100 ng) of linearized plasmid DNA with 40 μL of competent cells in an electroporation cuvette. Incubate for 2 minutes on ice.

2,  Electroporate samples using the following parameters:
(i) ECM 630 electroporator (BTX, San Diego, CA USA): cuvette gap, 2.0 mm, charging voltage, 1500 V; resistance, 200Ω; capacitance, 50 μF.

(ii) Gene Pulsar II electroporator (Bio-Rad Laboratories, Hercules, CA, USA); cuvette gap, 2.0 mm, charging voltage, 1500 V; resistance, 200Ω; capacitance, 25 μF.

3,  Immediately after electroporation, resuspend samples in 0.5 mL 1.0 M Sorbitol and 0.5 mL YPD. Incubate in a 30°C shaker for 1 hour and then plate 100-200 μL onto YPDS agar media with increasing concentrations of Zeocin (200 to 1000 μg/ml Zeocin) or 300 μg/ml Geneticin for selection of multi-copy integrants.

use two plates: 500 ug/ml, 1000 ug/ml

4, For higher colony numbers, after the outgrowth step, centrifuge the culture tubes at 1000 x g, pour off the supernatant, and resuspend the pellet in the remaining droplet of media. Divide the suspension between the two levels of antibiotic plates and spread the culture out onto the plate.

5, Incubate plates at 30°C for 2 to 3 days, until colonies are well formed. There may be some background growth visible, but definite colonies will also be present.

Transformation of Pichia by Electroporation

Day 1:
1. Streak each strain from a working glycerol stock or stab for single colonies on YPD agar plates. Grow at 24–30°C for 3–5 days until colonies form.
Day 2:
2. Inoculate 10 mL of YPD media in a sterile 125 mL baffled flask with a single colony from the YPD agar plate.
3. Incubate shaking at 300 rpm at 24–30°C for 1–2 days. This is your starter culture.

Note: It is important to have adequate aeration for growth. Always use at least a 1:5 ratio of media to flask volume.
4. Use the starter culture to inoculate 100 mL of YPD media in a sterile 1 liter flask to an OD600 of 0.2. Grow the culture for 1–2 days at 24–30°C.
Day 3 or 4:

5. Monitor the OD600 of the culture until it reaches between 1.3–1.5 (log phase growth). Transfer cells to a 300 mL centrifuge bottle, centrifuge at 1,500 × g at 4°C for 5 minutes. Resuspend the pellet with 250 mL of ice-cold, sterile water.

6. Centrifuge the cells as in previous step, then resuspend the pellet with 50 mL of ice-cold, sterile water.
7. Centrifuge the cells as in previous step, then resuspend the pellet in 10 mL of ice-cold 1 M sorbitol.
8. Centrifuge the cells as in previous step, then resuspend the pellet in 300 μL of ice-cold 1 M sorbitol for a final volume of approximately 500 μL. Keep the cells on ice and use that day. Do not store cells.

For each transformation (i.e., each strain and plasmid construct):
9. Mix 80 μl of the electrocompetent PichiaPink cells with 5–10 μg of linearized plasmid DNA and transfer to an ice-cold 0.2 cm electroporation cuvette.
10. Incubate the cuvette with the cells on ice for 5 minutes.
11. Pulse the cells in the electroporator
The time constant should be ~5 milliseconds.
12. Immediately after pulsing the cells, add 1 mL of ice-cold YPDS media to the cuvette and mix by pipetting up and down.
13. Incubate the cells at 24–30°C for at least 2 hours without shaking. Do not need to remove the cells from the cuvettes.
Note: the cells may be incubated for up to 12 hours.
14. After incubation, spread 100–300 μL of the cell mixtures on PAD selection plates, and incubate at 24–30°C for 3–10 days until distinct colonies are formed.
15. Pick 3–8 white colonies from each plate and restreak on fresh PAD selection plates. Proceed to small-scale expression to test for secreted expression of GOI.

Optional: for stable expression, analyze the integration of GOI insert in the transformed PichiaPink strain by PCR

The color of the colonies indicates the relative expression levels of protein of interest. The pink colonies express very little ADE2 gene product, while the white colonies express higher amounts of the ADE2 gene product, suggesting that those colonies have more copies of the integrated construct.

Materials:
• Working glycerol stock of PichiaPink Strains
• YPD media and YPD agar plates
• Sterile water, ice cold (place on ice the day of the experiment)
• 1 M sorbitol, sterile and ice cold (place on ice the day of the experiment)
• 125 mL and 1 liter baffled culture flasks, sterile
• 300 mL centrifuge bottles, sterile
• 5–10 μg pure PichiaPink plasmid construct (i.e., pPinkα-HC containing GOI insert) for each transformation
• YPDS media
• PAD selection plates
• Electroporation device and 0.2 cm cuvettes

Transformation of Pichia by Electroporation (condensed).

1. Grow a 5-mL overnight culture of Pichia pastoris cells in YPD in a 30°C shaking incubator.

2. The next day, dilute the overnight culture to an A600 of 0.15–0.20 in a volume of 50 mL YPD in a flask large enough to provide good aeration (Starting volumes can be scaled up or down.)

3. Grow yeast to an A600 of 0.8–1.0 in a 30°C shaking incubator. Based on a generation time of 100–120 min, yeast should reach 0.8–1.0 in 4
to 5 h.

4. Centrifuge the culture at 500× g for 5 min at room temperature and pour off the supernatant.

5. Resuspend the pellet in 9 mL of ice-cold BEDS solution [10 mM bicine-NaOH, pH 8.3, 3% (v/v) ethylene glycol, 5% (v/v) (dimethyl sulfoxide) DMSO, and 1 M sorbitol] supplemented with 1 mL 1.0 M dithiothreitol (DTT). Note that various concentrations (0–200 mM) of DTT were tested, but the amount used in this procedure (100 mM) yielded the most transformants.

6. Incubate the cell suspension for 5 min at 100 rpm in the 30°C shaking incubator.

7. Centrifuge the culture again at 500× g for 5 min at room temperature and resuspend the cells in 1 mL (0.02 volumes) of BEDS solution without DTT. We have also found transformation efficiency may be increased by resuspending cells in smaller volumes (0.005–0.01 volumes) of BEDS solution.

8. The competent cells are now ready for transformation. Alternatively, freeze cells slowly in small aliquots at −80°C by placing the aliquots inside a styrofoam box. Competent cells can be stored for at least 6 months at this temperature.

9. Mix approximately 4 μL (50–100 ng) of linearized plasmid DNA with 40 μL of competent cells in an electroporation cuvette. Incubate for 2
min on ice.

10. 1 Electroporate samples using the following parameters:

(i) ECM® 630 electroporator (BTX, San Diego, CA, USA):
cuvette gap, 2.0 mm;
charging voltage, 1500 V;
resistance, 200 Ω;
capacitance, 50 μF.

(ii) Gene Pulser® II electroporator (Bio-Rad Laboratories, Hercules, CA, USA):
cuvette gap, 2.0 mm;
charging voltage, 1500 V;
resistance, 200 Ω;
capacitance, 25 μF.

11 Immediately after electroporation, resuspend samples in 1 mL cold 1.0 M sorbitol and then plate on selective media (YNB, 2% dextrose + 1.0 M sorbitol) for auxotrophic strains.

Alternatively, if using zeocin-based plasmids, resuspend samples in 0.5 mL 1.0 M sorbitol and 0.5 mL YPD, incubate in a 30°C shaker for 1 h, and then plate on media containing increasing concentrations of zeocin (100, 250, 500, or 1000 μg/mL) for the selection of multicopy integrants. Note that increased numbers of transformants can be achieved for both types of selectable markers by incubating the resuspended cells in a 30°C shaker for longer periods of time (1–3 h). However, this is partly due to replication of transformants.

YPD media is composed of 1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) dextrose.

YNB media is composed of 0.17% (w/v) yeast nitrogen base without amino acids and 0.5% (w/v) ammonium sulfate.

Short Protocol

• Grow 50 mL cells to desired OD₆₀₀
• Centrifuge cells for 5 min 4000× g at 4°C
• Resuspend in 9 mL BEDS + 1 mL 1.0 M dithiothreitol (DTT)
• Incubate for 5 min with shaking
• Centrifuge cells for 5 min 4000× g at 4°C
  
• Resuspend cells in 1 mL BEDS
• Aliquot into individual 1.5 mL tubes
• Place in −80°C freezer until needed

Transformation

• Mix DNA with cells in cuvette
• Incubate for 2 min on ice
• Pulse
  
• Add recovery medium (optional incubation for 1–3 h)
• Plate

BEDS solution is composed of 10 mM bicine-NaOH, pH 8.3, 3% (v/v) ethylene glycol, 5% (v/v) dimethyl sulfoxide (DMSO), and 1 M sorbitol.
YPD media is composed of 1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) dextrose.

Transformation of Pichia by Electroporation (protocol 2).

Based on Invitrogen protocol

Materials

5-100 ug pure plasmid DNA containing gene of interest, linearized by restriction digestion.

Vector can be linearized with SacI, PmeI, and BstX.
Extra YPD media

YPDS plates (with proper antibiotic, ie, 100 and 500 ug/mL Zeocin)

500 mL YPD Media (in a 2L baffled flask)
1 L cold, sterile ddH2O
25 mL cold, sterile 1M sorbitol
Electroporation cuvettes (0.2 cm gap width)
Incubator/shaker set at NO HIGHER THAN 30 oC. If the incubator doesn’t keep consistent heat, set it at
28 oC.

A Pichia strain, streaked out on a YPD plate.
NOTE: Because this protocol is time-consuming, It is recommended that you do multiple transformation simultaneously
(ie, several different plasmids, or different amount of plasmid, etc). This will save you time in the end.

Protocol
1. Prepare DNA (purify linearized DNA and elute with water).

2. Pick a single colony of your Pichia strain to grow in 10 ml YPD overnight at 30 oC in a 125 ml sterile flask.
3. Innoculate 500 mL YPD in a 2 L flask with 0.1-0.5 ml of the overnight culture. Grow overnight again to an O.D. of 1.2-1.5.
4. Centrifuge the cells at 1500 x g for 5 minutes at 4 oC. Resuspend the pellet with 500 mL of ice-cold, sterile water.
5. Centrifuge the cells as in step 3, and then resuspend the pellets in 250 mL of ice-cold, sterile water.
6. Centrifuge the cells again, then resuspend in 20 ml of ice-cold 1M sorbitol. Transfer to a 50 ml conical tube.
7. Centrifuge the cells again, the resuspend the pellet inn 1 mL of ice-cold 1M sorbitol for a final volume of about 1.5 mL. Keep the cells on ice and use that day—do not store the cells!
8. Mix 40 μL of the cells from step 6 with linearized DNA prepared as per step 1, and then transfer to an ice-cold 0.2 cm electroporation cuvette. Be sure to tap the cells down to the bottom of the cuvette
9. Incubate the cuvette with the cells and DNA on ice for 5 minutes.
10. Pulse the cells:
Pichia: 25 uF, 200 ohm, 2000 V
11. Immediately add 1 ml of ice-cold 1M sorbitol to the cuvette. Transfer the cuvette contents to a sterile 15 ml tube.
12. Let the tube incubate at 30 oC without shaking for 1-2 hours.
13. Spread 10, 25, 50, 100, and 200 μL each on separate, labelled YPDS plates containing xx adenine
14. Incubate plates from 3-10 days at 30 oC until colonies form.

Transformation of Pichia by Electroporation (protocol 3).

Growth medium YPD (1% yeast extract, 2% bactopeptone, 2% dextrose)
Washing solution Ice-cold, sterile bidistilled water; ice-cold sterile 1 M sorbitol
Electroporation solution Ice-cold sterile 1 M sorbitol
Outgrowth medium Ice-cold sterile 1 M sorbitol
Outgrowth plates YPDS (1% yeast extract, 2% bactopeptone, 2% dextrose, 1 M sorbitol, 2 % agar)
Cuvette 2 mm gap width

Making electrocompetent cells:
1. Streak Pichia cells from a glycerol stock (30 % glycerol) onto YPD plates to grow single colonies.
2. Inoculate 10 ml YPD with one colony and grow 12-14 h with shaking at 30 °C to an O.D.600 of ~3.0. It is important to have healthy log phase cells.
3. Inoculate 250 ml YPD with an aliquot of the overnight culture to reach an O.D.600 of 0.005 and grow ~6 h to an O.D.600 of 1.0-1.3.
4. Harvest by centrifugation at 1,500 x g for 5 minutes at 4 °C.
5. Gently resuspend in 250 ml ice-cold ddH2O using a glass pipette, centrifuge as above.
6. Repeat washing step with 125 ml ice-cold ddH2O.
7. Resuspend in 20 ml ice-cold sorbitol and centrifuge as above. Resuspend in 0.5 ml of 1 M sorbitol and keep on ice.
Use the cells that day.
Electroporation of cells:
1. Add 10 μl DNA (5-10 μg) to 80 μl of electrocompetent cells. Gently mix with a blue-tip pipette several times. Transfer mixture into a prechilled cuvette and incubate on ice for 5 minutes.
2. Wipe moisture from the cuvette and insert the cuvette into the device.
3. Electroporation:
Mode Prokaryotes “¡“
Voltage (V) 1,500 V
Time constant (t) 5 ms
4. Immediately add 1 ml of ice-cold sorbitol to the cuvette. Transfer sample to sterile 15 ml tube and incubate for 2 hours without shaking at 30 °C.
5. Spread 200 μl aliquots on YPDS plates and incubate for 3 days at 30 °C until colonies appear. Number of colonies is increased about tenfold by adding 1 ml YPD and shaking for 3 hours before spreading thus incubating for a total of 5
hours.

Expected results:

Transformation efficiency up to 2 x 102 transformants/μg of DNA.

Pilot expression

The pilot experiments involve growing a small culture of each recombinant, taking time points, and analyzing the cell pellet and supernatant from each time point by SDS polyacrylamide gel electrophoresis (SDS-PAGE).

Pichia can be grown in tubes (15 mL or 50 mL conical tubes), multi-well plates, or small shake flasks if so desired.

Higher density formats such as 96-well plates can greatly simplify the testing of multiple strain, vector, and secretion signal combinations when using the PichiaPink Secretion Optimization Kit. Growth in 96-well plates is best done with a 3-mm throw shaker at a high agitation speed.

DNA2.0 Protocol (96 well plate)

Media

1M Potassium phosphate buffer, pH 5*, filter- sterilize, store at room temperature

• The pH may need to be different to suit the expressed protein. P. or K. pastoris will grow at pH 3 to pH 7. 0. A more acidic pH will inhibit many types of proteases.
  

134 g/L Yeast Nitrogen Base (without amino acids): filter- sterilize, store at 4°C

0.2 g/L Biotin: filter-sterilize, store at 4°C
40% glucose: autoclave at 121°C for 15 minutes, store at room temperature.

BMD1%: 200 mL of 1 M Potassium Phosphate buffer pH 5, 100 mL of 134 g/L Yeast Nitrogen base, 2 mL of 0.2 g/L Biotin, 27.5 ml of 40% glucose, dH20 to 1L. Filter sterilize, store at 4°C.

BMM2: 200 mL of 1 M Potassium Phosphate buffer pH 5, 100 mL of 134 g/L Yeast Nitrogen base, 2 mL of 0.2 g/L Biotin, 10 mL methanol, dH20 to 1L. Filter sterilize, store at 4°C.

BMM10: 200 mL of 1 M Potassium Phosphate buffer pH 5, 100 mL of 134 g/L Yeast Nitrogen base, 2 mL of 0.2 g/L Biotin, 50 mL methanol, dH20 to 1L. Filter sterilize, store at 4°C.

1. For each 96-well x 2 mL plate, prepare 30 mL of BMD1% containing 200 μg/ml Zeocin or 300 μg/ml Geneticin. Add 300 μL/well to the plate, except for 1 well used as a negative control culture.

2. Using sterile pipette tips, pick colonies from the transformation plates, preferably from the highest Zeocin concentration transformation plates first. Place the tips into the wells and twirl to mix the colony with the media. Temporarily, leave the tips in the plate. For the negative control well, inoculate from the P. or K.pastoris host strain plate.

3. When the plate has been filled with picked colonies, use an 8-channel pipette to remove the tips, swishing the media up and down a few times to dislodge any cells from the tips.

To make a clonal-retain archive plate, touch the pipette tips to the surface of a YPD + 200 μg/ml Zeocin plate or 300 μg/ml Geneticin. Discard the tips.
Incubate the archive plate at 30°C for a few days, then store at 4°C. This will provide a source of any productive clone.

4. Seal the 96-well culture block with a breathable membrane tape. Incubate for 60 hours at 28°C, shaking vigorously, to build biomass before induction.

5. Add 250 μL/well of BMM2 to begin induction. Continue incubation at 28°C for 12 hours.

6. Add 50 μL/well of BMM10 to continue production.

7. Boost the methanol content by adding 50 μL/well of BMM10 twice a day for about 48-60 hours.

Invitrogen Protocol (50 ml scale)

1. Using a single colony, inoculate 10 mL of BMGY medium in a 125 mL baffled flask. Grow the cells for 1–2 days at 24–30°C in a shaking incubator set to 250–300 rpm.
2. Transfer the cells to 50 mL conical tubes and centrifuge at 1,500× g for 5 minutes at room temperature. Decant the BMGY supernatant and resuspend the cell pellet in 1 mL of BMMY medium to induce expression.

Note: Make sure to replace the caps of the tubes with porous tape to maximize aeration.
3. The next day, remove 100 μL from the sample for gel analysis, and add 100 μL of 40% methanol.

4. Continue to grow the cells overnight in the 30°C shaking incubator set to 300 rpm.
5. Harvest the cells by centrifuging for 10 minutes at 1,500 × g.
6. Transfer the supernatant to a separate tube and store both the supernatant and the cell pellet at –80°C until ready to assay. Freeze quickly in liquid N2 or a dry ice/alcohol bath.
7. Return the cells in BMMY medium to the 30°C shaking incubator to continue growth overnight. Analyze the supernatants and cell pellets for protein expression by Coomassie or Silver-stained SDS-PAGE and western blot or functional assay.

Protocol by Day

Day 1-5:

streak on an YPD plate to grow single colonies at 29 oC. Select 5 colonies (about 1 mm in dia) for expressing level test.

Day 1
Starter culture: Culture a single transformed colony in 5 ml BMGY in a pathogenic free 50-ml conical tube at 29C at 250rpm shaking. Grow the cells for 1 day until it reaches OD600 of 2-6.

Day 2
-- When the culture reached OD₆₀₀ nm of approx 5, harvest the cells by centrifuging at 1500 g x 5 min at room temperature. Decant the BMGY (0.5% methanol) and add 1 ml of BMMY to the cell pellet. Replace the caps of the tubes by 4 layer of sterile gauze and culture the cells in the 50-ml conical cells at 29C with vigorous shaking (250 rpm).

Day 3 (induction Day 1)
-- Remove 100 ul of the culture and spin it to collect both supernatant and cell pellet and store them at -80C for SDS-PAGE analysis. Add 100 ul of 40% sterile methanol to the culture to continue inducing the protein expression under the same culture conditions. This is because the methanol evaporate during the overnight culture especially in this small volume culture.

Day 4 (induction Day 2)
-- Remove 100 ul of the culture and spin it to collect both supernatant and cell pellet and store them at -80C for SDS-PAGE analysis. Add 100 ul of 40% sterile methanol to the culture to continue inducing the protein expression under the same culture conditions.
Note: the 1 ml culture medium started to evaporation as its volume was reduced significantly

Day 5 (Induction Day 3)
-- The culture is about to dry out.

Media
BMGY or BMMY (Use endotoxin free water or ultrapure water):
YP concentrate: 700 ml containing 10 g yeast extract and 20 g peptone containing 100 mM potassium phosphate (autoclaved).
Add the following sterile solutions to the 700 ml YP:
100 ml of 10X YNB
20 ml of 50X Biotin (0.02% diluted from 100 mM (2.4% biotin stock)
100 ml of 10x glycerol stock (10%)
BMGY: add 100 ml of 10x Glycerol stock (10%) to final of 1% in the medium.
BMMY add 100 ml of 10x methanol stock (5%) to a final of 0.5%

Scale up protein expression in PichiaPink stains

1. Using a single colony, inoculate 25 mL of BMGY in a 250 mL baffled flask. Grow at 24–30°C in a shaking incubator (250–300 rpm) until culture reaches an OD600 of 2–6 (approximately 2–3 days).

2. Use this 25 mL culture to inoculate 1 liter of BMGY in a 3 or 4 liter baffled flask and grow at 24–30°C with vigorous shaking (250–300 rpm) until the culture reaches log phase growth (OD600 = 2–6).

3. Harvest the cells by centrifuging in sterile centrifuge bottles at 1,500–3,000 × g for 5 minutes at room temperature. To induce expression, decant the supernatant and resuspend the cell pellet in 200 mL of BMMY medium.

Note: The resuspension step above concentrates the culture 5-fold, thereby reducing the need for concentrating the secreted proteins if the expression level is not particularly high.

4. Aliquot the culture between two 1 liter baffled flasks. Cover the flasks with PureLink Air Porous Tape, 2 layers of sterile gauze, or
cheesecloth and return to incubator. Continue to grow at 24–30°C with shaking.

5. Add 100% methanol to 0.5% every 24 hours until the optimal time of induction is reached as determined from the time course study.

6. Harvest cells by centrifuging at 1,500–3,000 × g for 5 minutes at room temperature.

7. For intracellular expression, decant the supernatant. You can process the cells immediately or store at –80°C until ready for use.
8. For secreted expression, save the supernatant, chill to 4°C, and concentrate if desired (see below). Proceed directly to protein purification,
or store the supernatant at –80°C until ready to process further.

Protocol by Day

Day 1-3 or 1-4:

streak the transformed yeast stock on an YPDa plate to grow single colonies at 29C.

Day 4 or 5
Starter culture: Culture a single colony in 15 ml BMGY medium in a pathogenic free 125-ml baffled flask with pore cap at 29C at 250rpm shaking until culture reach an OD600 of 2-6 (approx 24 h or 2-3 days to OD600 of 4-5)

Day 6-7
Use the 15 ml of the starter culture to inoculate 500 ml BMGY in 2-liter pathogenic free baffled flask with pore caps to a final OD600 of 0.1-0.2. Grow at 29C with vigorous shaking (250-300 rpm) until the culture reaches log phase of OD600 2-6.

Day 8 (Induction Day1)
Harvest cells by centrifuging in sterile bottles (Beckman coulter) at 2,500 x g for 5 min at room temperature. Decant the supernatant and resuspend the cells in 100 ml BMMY (0.5% methanol) in a pathogenic free 500-ml flask with pore. Culture the cell at 29 oC and 250rpm shaking. (take 100 ul sample at zero time of induction for SDS-PAGE analysis)

Day 9 (Induction Day 2):

Add 0.6 ml 100% methanol (sterile using 0.22 um on a sterile 250-ml plastic filtration device) to the 100 ml culture in BMMY and continue the culture. (take 100 ul 24h sample for analysis)
Day 10 (Induction Day 3, 48 h)

Harvest the cells by centrifuging in sterile bottles  at 2,500 x g for 10 min at 4 oC. Collect the clear supernatant and frozen in liquid nitrogen, store at -80 oC. (take 100 ul supernatant for the analysis).

Media for protein expression under low endotoxin condition
BMGY or BMMY (Use endotoxin free water or ultrapure water to prepare):

YP solution:

700 ml containing

10 g yeast extract,

20 g peptone

100 mM potassium phosphate (autoclaved).

Add the following sterile solutions to the 700 ml YP:
100 ml of 10X YNB
100 ml of 10x glycerol stock (10%)
20 ml of 50X Biotin (0.02% diluted from 100 mM (2.4% biotin stock)
For BMGY: add 100 ml of 10x Glycerol stock (10%) to final of 1% in the medium.
For BMMY add 100 ml of 10x methanol stock (5%) to a final of 0.5%

Filter the media using pathogenic free filter (0.22 um) to further low endotoxin level before culture

Yeast glycerol stock strain preparation

PichiaPink strains: PPS1, S2, S3 and S4

Streak each strain on a YPDa agar plate and grow 3 days at 29C to obtain single colonies (most are white color colonies). All 4 strains are grown well after 3 days.

Preparation of stock strains:
Day 1
Starter culture: Culture two of a single colony of each strain in 10 ml YPDa medium in a 50-ml conical tube with cap (total 2 x 4) for 16-24 hours (starting at 10am) at 29C and shaking at 250 rpm.
(note: it is important to have the media to flask volume ratio: at least 1:5)

Day 2
Seed 200 ml YPDa with the starter culture to an OD600 of 0.2. Grow at shaking for 1-2days at 29C to an OD600 of 2-3.

Day 3
-- Harvest cells at 1500 x g for 5 min at 4C using 200-ml Beckaman coulter bottle (autoclaved). Alternatively, harvest cells by dividing 200 ml into 4 x 50-ml conical tube of pathogen free.
-- Resuspend the cells into YPDa containing 25% glycerol by adding to a final OD600 of 50-100. For 200 ml cell pellet, 4 ml of the YPD media is added and measure its OD at 600nm to an OD600nm of 90 as measured.

Media used for stock strain preparation
YPD: 1% yeast extract and 2% peptone (YP autoclaved) and 2% of dextrose (by adding 20% sterilized dextrose to the autoclaved YP medium

YPDa: Add 0.2% sterilized adenine (as 75X) to the YPD before culture (dilute 75x) in the YPD.

Invitrogen Protocol

To store cells for weeks to months, use YPD medium and YPD agar slants

1. Streak each strain to obtain single colonies on YPD agar plates. Grow 3–5 days at 24–30°C.
2. Transfer one colony to a YPD stab and grow for 3–5 days at 30°C.
3. You can store the cells on YPD for several weeks at 4°C.
To store cells for months to years, store frozen at –80°C.
Day 1:
1. Culture a single colony of each strain in 10 mL of YPD medium for 16–20 hours at 24–30°C, shaking at 300 rpm. This is your starter culture.
Note: It is important to have adequate aeration for growth. Always use 1:5 ratio of media to flask volume.
Day 2:
2. Seed 200 mL of YPD medium with the starter culture to an OD600 of 0.2. Grow shaking for 1–2 days at 24–30°C to an OD600 of 2–3.
Day 3 or 4:
3. Harvest the cells by centrifuging at 1,500 × g for 5 minutes. Remove the supernatant and resuspend the cells in YPD medium containing 25% glycerol to
a final OD600 of 50–100 (approximately 2.5–5.0 × 10⁹ cells/mL).
4. Aliquot the cells in cryovials (1 mL aliquots) and freeze in liquid nitrogen or a dry ice/ethanol bath and store at –80°C. Cells will be pink in color.

Chemical Yeast Transformation Protocol

Preparation:

1) Streak or patch yeast on YAPD for 24-48 hrs. Can also use yeast from older plates (3 months at 4 deg) if efficiency is not an issue and the strain cooperates.

2) Inoculate 2 x 3-5ml cultures in 2 x YAPD or SC media (15ml snap cap tubes) from the fresh streak and incubate O/N 30 deg, 200 rpm. SC media may need to be seeded more heavily (or more tubes may be required).

YAPD can be substituted for 2xYAPD but efficiency will drop some and yeast will take longer to grow.

Transformation:

1. Pre-warm 50 ml 2xYAPD to 30 deg in 500 ml flask (at least 10min). Turn on 42 oC water bath (takes 30 min or more).

2. Dilute O/N culture 1:10 in water and take an OD at 600nm. Use 2xYAPD (or YAPD or SC when appropriate. diluted 1:10 in water as a blank. The diluted culture will usually have an OD of between 0.1 and 1.0.

3. Dilute culture to approximately 0.1 OD in 50 ml pre-warmed 2xYAPD and incubate 3-6hrs until OD reaches 1.0 (or close). Yeast grown rates vary so be prepared to wait. It usually takes 5-6 hours but can take quite a bit longer.

EXAMPLE: The diluted culture in step 2 has an OD or 0.2 so the culture OD is 2.0. To get an OD of approximately 0.1 for step 3 you would add 2.5ml of culture to your pre-warmed 50ml 2xYAPD (20x dilution). Obviously its not exact because you will now have 52.5ml instead of 50ml but its close enough. If you want it exact you can simply remove 2.5ml 2xYAPD from the flask before you add your 2.5ml culture but its not necessary.

4. Spin at 3000 rpm, 10 min at RT.

5. While spinning yeast, boil salmon sperm DNA for 5 min with boiling cap and chill on ice. Use 3-4 times (storing at -20. and get a new tube. Do not boil over and over again.

6. Resuspend in 1 ml ddH2O and transfer to 1.5 ml tube.

7. Vortex 1 min or until yeast are in suspension (no clumps).

8. Pellet at max speed, 30sec, RT and resuspend in 1 ml ddH2O by vortexing as before. 100 ul will be used for each transformation so there are enough yeast for 10 transformations.

9. Transfer 100 ul of yeast to a separate 1.5 ml tube for each transformation and pellet at max speed, 30sec, RT and remove supernatant.

10. For each transformation make up the following (master mix or separately):

PEG 3500 w/v	240 ul
1.0 M LiAc	36 ul
2 mg/ml salmon sperm DNA	50 ul
plasmid(s). + water 1~5 ul of bacterial miniprep DNA	34 ul

360 ul TOTAL

11. Add the transformation mix(es. to the yeast pellets from step #9 and vortex 1 min or until yeast are in suspension (no clumps).

Optional: add 50 μl DMSO to each transformation and mix immediately.

12. Place yeast at 42 deg for 40min. For best efficiency the optimal shock time needs to be determined empirically and can vary from 10 min to 60 min. For simple single selection plasmid transformation 15-30 min usually works fine.

13. Pellet at max speed, 30 sec, RT and remove transformation mix.
Optional steps: 14 ~ 16

14. Add 500 ul 5 mM CaCl2 and resuspend cells thoroughly by gently pipeting up and down.

15. Let tubes stand for at least 5 min (but not longer than 15 min!). at room temperature.

16. Spin down cells at max speed, 30 sec, RT and remove liquid.

17. Add 100 ul ddH2O and use a 200 ul pipet tip to stir the cells into solution. If necessary, pipet the yeast up and down very gently.

18. Plate 100 ul on appropriate 10 cm selective plates and grow 2-4 days at 30 oC.

NOTES:  Efficiency varies greatly based on strain and transforming DNA. It can vary from 1×10^3 to 1×10^5 (or so) colonies per ug of input DNA (for plasmid DNA).

Media and Solutions:

YAPD (double for 2xYAPD):

10g of BactoYeast extract

20g of BactoPeptone

20g Dextrose

100mg adenine hemisulfate

q.s. to 1000ml with water.

Autoclave 20min on liquid cycle.

SC Medium:

Selective medium will vary based on strain and application.

Salmon sperm:

2mg/ml in TE. Sigma D1626. Make 100ml in a flask using a magnetic stirrer. Takes 2-4 hours to dissolve. Aliquot in 1.5ml tubes (1ml/tube).

PEG 3500 w/v:

Place 50gm of polyethylene glycol, MW 3350 (Sigma) in a 150 ml glass beaker and add 35 ml of ddH20. Stir for 1-2hrs with a magnetic stirring bar until dissolved. q.s. to 100 ml and keep stirring for at least 10min to mix. Filter using a 0.45 um filter and aliquot in 15ml conicals (10ml each) and store at RT.

LiAc:

1.0M Lithium Acetate. No need to pH.

Chemical Transformation Protocol 2

Reagents and Solutions

The following reagents and solutions are required for all four LiAc/SS-DNA/PEG protocols.
Lithium Acetate (1.0 M). Dissolve 5.1 g of lithium acetate dihydrate (Sigma Chemical Co. Ltd., St. Louis, MO) in 50 ml of water, sterilize by autoclaving for 15 min, and store at room temperature.

Polyethylene Glycol 3350 (50%, w/v). Dissolve 50 g of PEG 3350 (Sigma) in 30 ml of distilled, deionized water in a 150-ml beaker on a stirring hot plate. When the solution has cooled to room temperature, make the volume up to 100-ml in a 100-ml measuring cylinder and mix thoroughly by inversion. Pour the solution in to a suitable glass bottle and autoclave for 15 min. Store, securely capped, at room
temperature. Evaporation of water from the solution will increase the concentration of PEG and severely reduce the yield of transformants.
Single-Stranded Carrier DNA (2.0 mg/ml). Dissolve 200 mg of salmon sperm DNA (Sigma) in 100 ml of TE buffer (10 mM Tris-HCl, 1 mM Na2 EDTA, pH 8.0) by stirring at 4 ° for 1-2 hr. Store 1.0-ml samples at - 2 0 °. Denature the carrier DNA in a boiling water bath for 5 min and chill in ice/water before use. Boiled samples, stored at - 2 0 °, can be reused or reboiled three times without loss of activity.

Yeast Growth Media. Yeast strains are grown up on plates of YPAD agar (YpD13 supplemented with 100 mg adenine hemisulfate per liter). The yeast cells to be transformed are usually regrown in liquid 2× YPAD medium 3 (2% Bacto-yeast extract, 4% Bacto-peptone, 4% glucose and adenine hemisulfate, 100 mg/liter).

SC selection medium 12 is adjusted to pH 5.6 with 1.0 N NaOH and autoclaved. Commercial formulations of YPD agar (Bacto YPD agar) and broth (Bacto YPD broth) media are available from Becton Dickinson Microbiology Systems (Becton Dickinson, Sparks, MD; www.bd.com). These media should be supplemented with adenine hemisulfate as above.

Rapid Transformation Protocol

Day 1. Inoculate the yeast strain in a 2-cm 2 patch onto YPAD agar and incubate overnight at 30 °. Alternatively, the yeast strain can be inoculated into 5 ml of liquid medium (2× YPAD or SC selection medium) and incubated on a shaker at 30 ° and 200 rpm.

Day 2
1. Heat a tube of cartier DNA in a boiling water bath for 5 min and then chill in ice/water.

2. Scrape a 50ul portion of yeast from the YPAD plate and suspend the cells in 1 ml of sterile water in a 1.5 ml microcentrifuge tube. The suspension will contain about 5 x 108 cells. Cells grown overnight in 2× YPAD broth will reach a titer between 1 and 2 x 108/ml; the titer in SC medium will be about 5 × 107/ml. Harvest 2 ml of a YPAD culture and 5 ml of an SC
culture. Note: Cells in log phase growth on agar or in liquid medium will transform with high efficiency.

3. Pellet the cells at 13,000 rpm at room temperature in a microcentrifuge for 30 sec and discard the supernatant.
4. Add the following components of the Transformation Mix (T Mix) to the cell pellet in the order listed:

Component	Volume (ul)
PEG 3350 (50%, w/v)	240
Lithium acetate 1.0 M	36
Boiled SS-Carrier DNA (2 mg/ml)	50
Plasmid DNA (0.1 to 1/ug) plus water	34
Total volume	360

   
 
Be sure to vortex mix the carrier DNA before pipetting it.

5. Incubate the tube in a water bath at 42 ° for 40 to 60 min. Many laboratory strains will yield up to 1 x 105 transformants/ug plasmid after 60 min incubation. Extending the time at 42 ° to 180 min increases the yield to >1 x 10⁶/ug with some strains.

6. Microcentrifuge at 13,000 rpm at room temperature for 30 sec and remove the T Mix with a micropipettor.
7. Pipette 1.0 ml of sterile water into the tube and resuspend the cells by stirring with a micropipette tip and then vortex mixing vigorously.
8. Pipette 10- and 100ul samples onto plates of appropriate SC selection medium, incubate at 30 ° for 3 - 4 days, and isolate transformants. The 10-/ul
samples should be pipetted into 100-#1 puddles of sterile water.

This protocol can be used with cultures that have been stored at room temperature or in a refrigerator. The yield will be reduced with older cultures but will generally be sufficient to isolate a number of transformants of the desired genotype.

Rapid Transformation with yeast grown on plate

1. Prepare a transformation master mix. The following ingredients provide enough reagents forfive transformation reactions. Combine in a microcentrifuge tube:

100 μL sterile 2 M lithium acetate (freshly prepared)
400 μL sterile 50% PEG-3350
4 μL 2-mercaptoethanol (STINKY!! add this in the fume hood!)

Set up individual transformation reactions - for each transformation:

2. Add 15 μL of the denatured salmon sperm DNA (2 mg/mL) to labeled microcentrifuge tubes.

Note: It is important for the salmon sperm DNA to be single-stranded for this procedure to work well. Boil the DNA for 5 minutes to denature the DNA. Quick chill the DNA by placing it immediately on ice. Keep the DNA on ice until you are ready to use it.

3. Add 5 μL of miniprep plasmid DNA to the appropriately labeled microcentrifuge tube.

4. Add 100 μL of transformation mix from step 1 to the microcentrifuge tube. Vortex for 10-15 seconds to mix the contents.

5. Using a sterile toothpick, scrape a large yeast colony from a YPD plate. Transfer the yeast to the microcentrifuge tube containing the transformation/DNA solution (Step 4) by twirling the toothpick several times. Be sure that the cells are uniformly suspended before proceeding.

6. Incubate the transformation mixtures at 37 ̊C with shaking for 30-45 min. Plate the transformed cells on selective media (zeocin)

High-Efficiency Transformation Protocol

This protocol can be used to generate sufficient transformants in a single reaction to screen multiple yeast genome equivalents for plasmids that complement a specific mutation. It can also be used to transform integrating plasmids, DNA fragments and oligonucleotides TM for yeast genome manipulation. Finally, it is used to optimize the conditions for transformation of a particular yeast strain, for example, the transformation of a plasmid library into a two-hybrid yeast strain transformed with a bait plasmid by the rapid transformation protocol. The high efficiency protocol can also be employed to transform a yeast strain simultaneously with two different plasmids, such as the two-hybrid bait and prey plasmids.

Day 1. Inoculate the yeast strain into 5 ml of liquid medium (2x YPAD or SC selection medium) and incubate overnight on a rotary shaker at 200 rpm and 30 °. Place a bottle of 2x YPAD and a 250-ml culture flask in the incubator as well.

Day 2
1. Determine the titer of the yeast culture by pipetting 10 ul of cells into 1.0 ml of water in a spectrophotometer cuvette and measuring the OD at 600 nm. For many yeast strains a suspension containing 1 x 10⁶ cells/ml will give an OD600 of 1.0. Alternatively, titer the culture using a hemocytometer.

2. Transfer 50 ml of the prewarmed 2x YPAD to the prewarmed culture flask and add 2.5 x 10⁸ cells to give 5 x 10⁶ cells/ml.

3. Incubate the flask on a rotary or reciprocating shaker at 30 ° and 200 rpm.

4. When the cell titer is at least 2 × 10⁷ cells/ml, which should take about 4 hr, harvest the cells by centrifugation at 3000g at room temperature for 5 min, wash the cells in 25 ml of sterile water and resuspend in 1 ml of  sterile water.

Boil a 1.0-ml sample of cartier DNA for 5 min and chill in an ice/water bath while harvesting the cells. Transfer the cell suspension to a 1.5-ml microcentrifuge tube, centrifuge for 30 sec, and discard the supernatant.

Add water to a final volume of 1.0 ml and vortex-mix vigorously to resuspend the cells.

Pipette 100 ul samples (ca. 10⁸ cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at 13,000 rpm at room temperature for 30 sec, and remove the supematant.

Make up sufficient T Mix for the planned number of transformations plus one extra. Keep the T Mix in ice/water.

	Number of transformations planned
Reagents	1	5 (6x)	10 (11x)
PEG 3350 (50%, w/v)	240 ul	1440ul	2640ul
Lithium acetate 1.0 M	36ul	216 ul	396ul
Boiled SS-CarrierDNA (2 mg/ml)	50ul	300 u1	550ul
Plasmid DNA plus water	34ul	204ul	374ul
Total volume	360ul	2160 ul	3960 ul

  

Add 360ul of T Mix to each transformation tube and resuspend the cells by vortex mixing vigorously.
11. Incubate the tubes in a 42 ° water bath for 40 min.

12. Microcentrifuge at 13,000 rpm at room temperature for 30 sec and remove the T Mix with a micropipettor.

13. Pipette 1.0 ml of sterile water into each tube; stir the pellet with a micropipette tip and vortex vigorously.

14. Plate appropriate dilutions of the cell suspension onto SC selection medium.

For transformation with an integrating plasmid (YIp), linear construct, or oligonucleotide, plate 200ul onto each of five plates; for a YEp, YRp, or YCp library plasmid dilute 10ul of the suspension into 1.0 ml of water and plate 10- and 100ul samples onto two plates each. The 10ul samples should be pipetted directly into 100ul puddles of sterile water on the SC
selection medium.

15. Incubate the plates at 30 ° for 3 to 4 days and count the number of transformants.

Making Yeast Competent Cells and Yeast Cell Transformation

Disposables and Reagents

1. Bacto-Yeast Extract (Fisher)

2. Bacto-Peptone (Fisher)

3. Glucose (Dextrose) (Fisher)

4. Bacto-agar (Fisher)

5. Deionized H2O

6. Glycerol (Sigma)

7. DMSO (Dimethyl sulfoxide) (Sigma)

8. 1.5 ml centrifuge tubes (appendorf)

9. PEG 3350 (Sigma)

10. LiAc (Lithium acetate dihydrate) (Sigma)

11. Salmon sperm DNA (Invitrogen)

12. YNB (Yeast Nitrogen Base) (Sigma)

13. MES (2-(N-morpholino)ethanesulfonic acid)

14. ADE (Sigma)

15. Glucose

Equipments

1. Water bath (VWR)

2. Centrifuge (Eppendorf)

3. 30 °C shaker and incubator (VWR)

4. Standard petri dishes (VWR)

Procedure

1. Make yeast competent cells (Modified from Gietz & Schiestl, 2007)

1) Get the yeast strains and streak on YPAD plates. Let cells grow 2 d before inoculation.

2) 1st Inoculation: inoculate one colony into 25 ml YPAD liquid medium. Grow cells overnight at 30 °C with shaking speed around 200 rpm.

3) 2nd inoculation. Transfer the 25 ml cell culture into 75 ml YPAD medium. Grow cells at 30 oC for 4 h.

4) Harvest cells by centrifugation at 3000 g for 5 minutes, wash cells with 0.5 volumes of sterile H2O.

5) Centrifuge again with 3000 g for 5 minutes.

6) Re-suspend cells in 0.01 volumes of sterile H2O, transfer to a suitable centrifuge tube and pellet at 3000 g for 5 min at 20 oC.

7) Re-suspend cell pellet in 0.01 volumes of filter sterilized frozen competent cell solution (5 % v/v glycerol, 10% v/v DMSO).

8) Dispense 50 ul cells into 1.5 ml appendorf tubes.

9) Place the tubes into box with Styrofoam or cardboard. (Slowly freezing is essential for good survival rates).

10) Store the box into -80 oC freezer. (Cells can be kept at this condition for up to one year).

2. yeast cell transformation

11) If use competent cells stored at -80 oC, thaw cells at 37 oC water bath for 15-30 S. if use freshly made competent cells, go to step 2).

12) Centrifuge at 13,000 g for 2 minutes to remove the supernatant.

13) Make the transformation solution for the planned number of transformation plus one extra (negative control) (see the recipes).

14) Add the solution to the cell pellet, votex to re-suspend the cells.

15) Incubate in a 42 oC water bath for 40 minutes.

16) Centrifuge at 13,000 g for 30 seconds and remove the supernatant.

17) Pipette 1 ml of sterile H2O into the transformation tube to re-suspend the pellet.

18) Plate 200 ul of the cell suspension onto the YNB+MA plate growth plate with your selection marker.

19) Incubate plates at 30 oC for 2~4 d.

Recipes

1. Make YPAD plate & liquid medium:

1% Bacto-Yeast Extract – 10 g/L

2% Bacto-Peptone – 20 g/L

2% Glucose (Dextrose) – 20 g/L

If making YPD plates, add 20 g Bacto-agar.

Fill up to 1 liter* with deionized HOH.

NO pH adjustments

(Note: autoclave agar & glucose separately, to avoid caramelization)

2. Transformation solution:

PEG 3350 (50 % (w/v)): 260 μl

LiAc 1 M: 36 μl

Salmon sperm DNA (10mg/ml): 10 μl

Plasmid: 3 μl

Sterile H2O: 51 μl

Total: 360 μl

3. YNB+ MA plate (100 ml):

YNB: 0.67 g

MES (20 mM): 0.39 g

ADE: 0.01 g

Agar: 2 g

Glucose: 2 g

References

1. Gietz R.D., Schiestl R.H. (2007). Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1): 1-4.

2. Lu Y., Chanroj S., Zulkifli L., Johnson M.A., Uozumi N., Cheung A., Sze H. (2011). Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23(1): 81-93.

Recipes

20% Dextrose
(10X)
To prepare 1 liter of 20% Dextrose (10X) stock solution:
1. Dissolve the contents of the Dextrose pouch from the PichiaPink Media Kit in 1000 mL of distilled water.
2. Autoclave for 15 minutes or filter sterilize.
Store at room temperature. The shelf life of this solution is approximately one year.

10X YNB (13.4% Yeast Nitrogen Base with Ammonium Sulfate, without amino acids)
Dissolve 134 g of yeast nitrogen base (YNB) with ammonium sulfate and without amino acids in 1000 mL of water and filter sterilize. Heat the solution to dissolve YNB completely in water. Store at 4°C. Alternatively, use 34 g of YNB without ammonium sulfate and amino acids and 100 g of ammonium sulfate. The shelf life of this solution is approximately one year. If you are using the YNB pouch included in the kit, follow the directions on the pouch.
Note: Pichia cells exhibit optimal growth with higher YNB concentrations, therefore, the amount of YNB used in this kit is twice as concentrated as YNB formulations for Saccharomyces.

50X B (0.02% Biotin)
Dissolve 20 mg biotin in 100 mL of water and filter sterilize. Store at 4°C. The shelf life of this solution is approximately one year.

100X A (0.2% Adenine)
Dissolve 200 mg of L-adenine in 100 mL of water. Heat the solution, if necessary, to no greater than 50°C in order to dissolve. Filter sterilize and store at 4°C. The shelf life of this solution is approximately one year.

10X D (20% Dextrose)
Dissolve 200 g of D-glucose in 1000 mL of water. Autoclave for 15 minutes, or filter sterilize. The shelf life of this solution is approximately one year.

10X M (5% Methanol)
Mix 5 mL of methanol with 95 mL of water. Filter sterilize and store at 4°C. The shelf life of this solution is approximately two months.

10X GY (10% Glycerol)
Mix 100 mL of glycerol with 900 mL of water. Filter sterilize or autoclave. Store at room temperature. The shelf life of this solution is greater than one year.

1 M potassium phosphate buffer, pH 6.0:
Combine 132 mL of 1 M K₂HPO₄, 868 mL of 1 M KH₂PO₄ and confirm that the pH is 6.0 ± 0.1 (if the pH needs to be adjusted, use phosphoric acid or KOH). Sterilize by autoclaving and store at room temperature. The shelf life of this solution is greater than one year.

Buffered Glycerol-complex Medium (BMGY) and Buffered Methanol-complex Medium (BMMY)
(1 liter)
1% yeast extract
2% peptone
100 mM potassium phosphate, pH 6.0
1.34% YNB
0.0004% biotin
1% glycerol or 0.5% methanol
1. Dissolve 10 g of yeast extract, 20 g peptone in 700 mL water.
2. Autoclave 20 minutes on liquid cycle.
3. Cool to room temperature, then add the following and mix well:
100 mL 1 M potassium phosphate buffer, pH 6.0
100 mL 10X YNB
20 mL 50X B
100 mL 10X GY
4.
For BMMY, add 100 mL 10X M instead of glycerol.
Store media at 4°C. The shelf life of this solution is approximately two months

Minimal Glycerol Medium ± Adenine (1 liter)
1.34% YNB
1% glycerol
0.0004% biotin
± 0.002% Adenine
1. Combine aseptically 800 mL autoclaved water with 100 mL of 10X YNB, 20 mL of 50X B, and 100 mL of 10X GY.
2. For growth of ade2 strains in this medium, a version can be made that contains adenine (called MGYA) by adding 10 mL of 100X A stock solution.
Store at 4°C. The shelf life of this solution is approximately two months.