Editing Pichia EXpression (section)

=== DNA2.0 Protocol (96 well plate)<br>  ===

<u>'''Media'''</u><br> 

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

*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.<br>

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

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

<br>'''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.<br> 

'''<br>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.<br> 

<br>'''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.<br> 

<br> 

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.<br> 

<br>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.<br> 

<br>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.<br> 

<br>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.<br>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.<br> 

<br>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.<br> 

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

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

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