Team:British Columbia/Protocols/Gcms

GC-MS Sample Preparation Protocol for yeast

Supplies Needed:

1. Erlenmeyer Flasks
2. Appropriate Media [Glucose (GPD) or Galactose (GAL)]
3. Spectrophotometer
4. sdH2O
5. Falcon Tubes
6. GLASS test tubes (large and small)
7. Pentane
8. Glass Beads
9. Sodium Sulphate (anhydrous)
10. GC Vials
11. 5mL Pasteur Pipets and Bulbs

Steps:

Part 1: Preparation for Extraction

1. Make 5mL overnight cultures of yeasts to be sampled (18-20 hours@ 30 degrees). The media used here must be SD-Leu (glucose).
2. Measure OD600 of the cultures using the spectrophotometer. The OD600 should be around 2.
3. Add the remaining cultures to Erlenmeyer flasks and dilute with 50mL of of SD-Leu.
4. Grow up the cultures for about 2-3 hours (until OD600 is 0.6-0.8).
5. Transfer all of the cultures into falcon tubes and spin down the cells (5 min@ 2500g).
6. Pour out media and resuspend the yeast pellets with about 47mL of SD-Leu (GPD) or SG-Leu (GAL).
7. Transfer the cultures to Erlenmeyer flasks and grow up overnight (18-20 hours@ 30 degrees).
8. Harvest cells (step 5).
9. Weigh the large glass tubes and record values.
10. Keep 1mL of the media and transfer to large glass tubes and discard the rest of the media.
11. Wash the yeast pellet with 5mL of sdH2O.
12. Transfer the 5mL of yeast to the glass tubes.
13. Spin down the cells (5 min @ 2500g).
14. Pour out water and weigh the glass tubes again to obtain the weight of the yeast pellet.
15. Proceed to Extraction.

Part 2: Extraction (all of the following steps should be done in the fumehood in Bohlmann lab)

1. Pipet in 2mL of pentane to each glass tube.
2. Add 0.5-1mL of glass beads to each tube.
3. Vortex as fast as possible without spilling for about 20 seconds.
4. Pour solvent into smaller glass tube. Pipet the solvent out for the media samples using the pasteur pipets. Avoid glass beads!
5. Add about 200uL of Sodium Sulphate to the small glass tubes.
6. Make one blank (4mL of pentane and Sodium Sulphate).
7. Repeat 1-4.
8. Bring down the volume to about 1mL by applying a stream of nitrogen.
9. Place GC vials in a rack and label them.
10. Pipet out solvent and transfer to vials.
11. Apply nitrogen to vials and bring down the volume to about 0.4mL.
12. Add pentane to 0.5mL line.

Protocol for bacteria

Protein expression of limonene synthase

1. 1. Grow 5 ml culture in LB overnight with appropriate antibiotics
2. 2. Pour 500 ml of 'terrific broth' into a 2 litre flask
3. 3. Add appropriate antibiotics
4. 4. Pipette 1 ml of culture into 500 mL
5. 5. Grow to OD600 = 0.8
6. 6. Add IPTG to final concentration of 1mM to 500 mL flask
7. 7. Induce expression at 16 degrees at 200 rpm
8. 8. Induce expression over night
9. 10. Spin down cells in centrifuge tubes, discard supernatant
10. 11. Store pellets at -80

Extraction and preparation

1. 1. Prepare assay buffer (see below)
2. 2. Re-suspend 1-2 grams of pellet per 4 ml of assay buffer in a falcon tube
3. 3. Sonicate cells until pellet turns translucent
4. 4. Spin down cells at max speed
5. 5. Pipette out supernatant (contains the limonene synthase) into another falcon tube
6. 6. Discard cell pellets
7. 7. Take 2 ml of supernatant and pipette into a different tube
8. 8. Add geranyl diphosphate (GPP) to a final concentration of 20 uM to the supernatant
9. 9. Add 1 ml of pentane (with isobutylbenzene) to the supernatant
10. 10. Incubate at 30 degrees for 1 hr
11. 11. Pipette out pentane layer into a glass tube with a pasteur pipette
12. 12. Add another 2 ml of pentane to supernatant
13. 13. Pipette out the 2 ml of pentane into the glass tube with a pasteur pipette
14. 14. Evaporate the total pentane to a final volume of 500 uL using nitrogen gas
15. 15. Pipette 500 ul into an agilent vial
16. 16. Send to Lina