Editing Pichia EXpression (section)

== <br>Transformation of Pichia by Electroporation<br>  ==

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Day 1:<br>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.<br>Day 2:<br>2. Inoculate 10 mL of YPD media in a sterile 125 mL baffled flask with a single colony from the YPD agar plate.<br>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.<br>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.<br>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.<br>7. Centrifuge the cells as in previous step, then resuspend the pellet in 10 mL of ice-cold 1 M sorbitol.<br>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. 

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For each transformation (i.e., each strain and plasmid construct):<br>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.<br>10. Incubate the cuvette with the cells on ice for 5 minutes.<br>11. Pulse the cells in the electroporator <br>The time constant should be ~5 milliseconds.<br>12. Immediately after pulsing the cells, add 1 mL of ice-cold YPDS media to the cuvette and mix by pipetting up and down.<br>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.<br>Note: the cells may be incubated for up to 12 hours.<br>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.<br>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<br> 

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.<br><br> 

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