Editing Pichia EXpression (section)

== 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. 

<br> 

=== 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> 

=== <br>  ===

=== Invitrogen Protocol (50 ml scale)<br>  ===

<br> 

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.<br>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.<br>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.<br>5. Harvest the cells by centrifuging for 10 minutes at 1,500 × g.<br>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.<br>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.<br><br> 

<br>'''Protocol by Day'''<br> 

'''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'''<br>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. 

<br>'''Day 2 '''<br>-- When the culture reached OD<sub>600</sub> 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). 

<br>'''Day 3''' (induction Day 1)<br>-- 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. 

<br>'''Day 4''' (induction Day 2)<br>-- 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. <br>Note: the 1 ml culture medium started to evaporation as its volume was reduced significantly 

<br>'''Day 5''' (Induction Day 3)<br>-- The culture is about to dry out. 

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