Editing Pichia EXpression (section)

== Transformation of Pichia by Electroporation (condensed).<br>  ==

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<br> 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<br> min on ice. 

10. 1 Electroporate samples using the following parameters: 

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

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

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Short Protocol<br> 

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*Grow 50 mL cells to desired OD<sub>600</sub> 
*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 <br> 
*Resuspend cells in 1 mL BEDS 
*Aliquot into individual 1.5 mL tubes 
*Place in −80°C freezer until needed

'''Transformation'''<br> 

*Mix DNA with cells in cuvette 
*Incubate for 2 min on ice 
*Pulse <br> 
*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. <br>'''YPD media''' is composed of 1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) dextrose.