Aim
The aim of this experiment is to identify lipid composition of
Y.lipolytica Po1f strain and compare DHA amount of engineered yeast cells with not engineered yeast cells using GC-MS analysis.
Experimental Design
Materials - lipid extraction
- Chloroform
- Methanol
- NaCl
- Anhydrous sodium sulfate (optional)
Lipid extraction
- Harvest the cell: 15 mL cell culture, 12000g, 5min
- Add 4 mL chloroform, 8 mL methanol, and 3.2 mL of 1% NaCl to the pellet, vortex after every addition.
- Agitate on a tube rotator at 30 rpm overnight.
- Add 4 mL chloroform and 4 mL of 1% NaCl, invert 30 times.
- Centrifuge 4000 g, 5 min, collect bottom layer.
- Blowdry (gentle stream of nitrogen) and dissolve the lipid in 1mL of a chloroform:methanol mixture (2:1, v/v).
- For N2 blowdry: it might be better to not expose DHA in air for it is relatively easy to be oxidized. CO2 is also ok, but if we do not have the equipment we can try airdry.
- Maybe anhydrous sodium sulfate can also be used to remove the moisture, but we have to use airdry to remove chloroform & methanol.
- Samples are stored at or below -60 ºC until ready for extraction to minimize oxidation and degradation of samples.
Materials - FAME (Fatty Acid Methyl Ester)
- 0.5M Methanolic KOH
- 10% BF3 in methanol
- DI H2O
- Hexane
FAME
- Two milligrams of the lipids are reconstituted in 1 mL of 0.5 M methanolic KOH and hydrolyzed at 80°C(60) for 40 mins
- Add 1 mL of fresh 10% BF3 in methanol. Transesterification is performed at 80°C for 40 min.
- Keep the temperature same all the time
- After transesterification, 2 mL DI H2O and 1 mL hexane are added to the sample to quench the reaction.
- The recovered organic phase is pooled and spiked with the methyl ester of 21:0 to a final concentration of 25.0 µg/mL.
GC-MS
- Sample preparation for testing GC-MS
- Standard: Pure DHA
- need to know the exact amount of DHA
- Blank: Lipid from Y.lipolytica (without engineering)
- Spiking: Lipid from engineered Y.lipolytica + DHA
- need to know the exact amount of DHA added
- Target: Lipid from engineered Y.lipolytica
- 1mL of samples are measured with GC-MS (Varian CP-3800 GC equipped with a Varian Saturn 2200 MS) to confirm peaks.
- The same measurement conditions as for GC-FID are used. The mass range is 50-400 m/z, scan time 0.5 sec/scan and emission current of 10uA.
- Acquisition is from 8 to 40 min.
- Compound retention times are listed in Table IV in the FAME doc
Expected
Compare to normal Y.lipolytica, engineered Y.lipolytica has higher DHA amount (30%)
Actual & Results
Results
- Observation of the recovery from the spike
- Recovery of 10010 → good
- If falls out of that range, other factors are affecting the result
- < 90: maybe smth like enzymes digested DHA - Kill the enzymes by heating
- > 100: maybe other components similar to DHA were present
Aim
The aim of this experiment is to assess the expression of each subunit of PUFA synthase.
Experimental Design
Each subunit of PUFA synthase is attached to a His-tag, so we use a His-tag antibody as
our primary antibody. An anti-Rabbit/Mouse IgG antibody is used as our secondary antibody.
Materials
- Y. lipolytica overnight culture
- Double-distilled H2O
- 0.2 M NaOH
- 2x SDS Sample Buffer
- Reagents for protein gel
- Transfer buffer
- Blocking buffer
- Wash buffer
- PVDF membrane
- PBS
- Primary antibody
- Secondary antibody
Protein Sample Preparation
- Harvest overnight yeast culture in YPD by centrifuging at 12,000 x g for 5 min at room temperature.
- Resuspend yeast cells in 100μL of double-distilled H2O.
- Add 100μL of 0.2 M NaOH.
- Incubate at room temperature for 5 min.
- Pellet yeast cells by centrifuging at 12,000 x g for 5 min at room temperature.
- Discard the supernatant.
- Resuspend yeast cells in 50μL of SDS Sample Buffer (2x).
- Incubate at 100°C for 3 min.
- Pellet yeast cells by centrifuging at 12,000 x g for 5 min.
- Transfer supernatant to a new 1.5mL centrifuge tube. Discard the pellet.
SDS-PAGE
- Load 15-20μL supernatant per well on a protein gel.
- Load 10μL of protein ladder.
- Run protein gel at 200V for 1.5 hr.
Western Blot
- Place the protein gel in transfer buffer.
- Soak the filter paper and PVDF membrane in the transfer buffer.
- Assemble the “transfer sandwich”.
- Transfer separated proteins to the PVDF membrane by electroblotting at 20V for 20 min.
- Incubate the membrane in PBS for 10 min.
- Block the membrane by adding blocking buffer and incubate at 4°C overnight on a shaker.
- Wash the membrane in PBS for 5 min.
- Add primary antibody (anti-His-tag) and incubate for 1 hr at room temperature.
- Wash the membrane in wash buffer for 10 min 3 times.
- Add secondary antibody and incubate for 1 hr at room temperature.
- Wash the membrane in wash buffer for 10 min 3 times.
- Add substrate to develop the western.
- Use ChemiDoc for chemiluminescent detection of the membrane.
Expected
Need to confirm size of each subunit
Actual & Results
Reference
Nevada. (2014). Western Blot to Examine Yeast Strains for Protein Expression. iGEM. https://static.igem.org/mediawiki/2014/5/52/UNR_Western_Blot_Protocol_to_detect_GFP_in_yeast.pdf
