Construct engineered Saccharomyces cerevisiae
Preparation of Saccharomyces cerevisiae receptor cells
- Pick a fresh Saccharomyces cerevisiae EBY100 monoclonal from YPD plates into 10 mL YPD liquid medium and
incubate at 30°C, 250 rpm overnight.
- Determine the OD600 value of the overnight culture to be between 3.0 and 5.0.
- Dilute 10 mL of YPD overnight culture to an OD600 value of 0.2~0.4.
- Continue to incubate in a shaker at 28~30°C for 3~6 hr to reach an OD600 value of 0.6~1.0.
- Centrifuge the yeast cells for 5 min at 1500 g at room temperature and discard the supernatant.
- Wash yeast cells with 10 mL of washing solution, followed by centrifugation at 1500 g for 5 min at room
temperature to collect the cells, and discard the supernatant.
- Resuspend yeast cells with 1 mL TE/LiAc in 50 μL portions per tube.
Make LB and YEDP media:
- Take a small amount of yeast strain Y2H frozen stock and incubate it in line on YPDA plate medium,
inverted at 30°C.
- Pick a single colony of yeast in a 2.0 mL centrifuge tube, add 1 ml of YPD liquid medium and shake
vigorously to disperse the clumps.
- Transfer 1:100 ratio to the appropriate amount of YPD liquid medium, incubate at 30°C, 220 rpm for 18-24
h, until stable, OD600>1.5.
- Transfer the culture to YPD liquid medium at a ratio of 1:4 and incubate at 30°C, 220 rpm for 3 h until
OD600=0.2-0.3.
- Transfer the yeast culture to a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 1 min at room
temperature, and discard the supernatant.
- Add 1 mL of TE buffer, re-suspend the precipitate, centrifuge at 12000 rpm for 30 s, and discard the
supernatant.
- Add 0.1 mL of TE-LiAc solution and re-suspend the precipitate.
Transformation of Saccharomyces cerevisiae cells
- Take 50 µL of receptor cells, add 2 µL of each plasmid to be transformed, and mix well
- Add 500 µL of transformation solution (PEG/LiAc, dimethyl sulfoxide), bounce on the wall of the tube and
mix well.
- Incubate in a shaker at 30°C for 1 hour, and mix by bouncing the wall of the tube at 15-minute
intervals.
- Add 1 mL of YPD culture solution and incubate for 1 hour at 30℃ in a shaker.
- Centrifuge at 3500 g for 5 minutes, leave the sediment and discard the supernatant.
- Re-suspend the precipitate with 150 μL TE, apply the corresponding SD plate and place in a 37℃ incubator
for 30-60 minutes until the surface liquid has penetrated into the medium, then invert the plate and place
in a 37℃ incubator overnight.
Verification of grown yeast cells using PCR and electrophoresis
- Jump out the cultured yeast from the YPD plate and place in YPD solution.
- Use a shaking incubator at 37℃ for 2-3 hours to grow more yeast cells.
- PCR after taking out of the incubator.
- 1 uL DNA, 5 uL Mix, 2 uL forward primer, 2 uL reverse primer, 10 uL ddH2O.
- Place on a shaker and beat well.
- Centrifuge in a centrifuge for 1s.
- put into thermal cycler for DNA replication.
- Prepare solution: TE buffer, and agarose gel.
- ddH2O is added to the TE buffer solution, and the solution is then used as a solvent to melt the agar.
- After adding agar, microwave for one minute.
- Load the PCR mixture into the agarose gel for electrophoresis: total left to right Marker, FAS1, FAS2,
ACC1.
- Finally check the results to determine if the desired yeast cells are cultured.