UW Stout/Sucrose Fermentation SP22

Introduction

The purpose of this protocol is to test if knockout yeast strains undergo fermentation, and if they do how much ethanol is produced. This will tell us if the gene that was knocked out plays a role in fermentation.

Materials

• 1 ml Yeast Culture (Approximately 1 million cells)
  • You may need to dilute with sterile H2O to approximate the 1 million cells. Keep total volume at 1 ml.
• PBS Buffer Solution
• Yeast Growth Solution
  • 0.85g YNB, 2.5 Ammonium Sulfate, 0.32g CSM, 5ml 100x Leucine, 100x Histine, 100x Uracil, 435 ml DI H2O
• 40% Sucrose Solution

Equipment

• High Performance Laser Chromatography (HPLC)
• Incubator
  • Set to 30°C
• Centrifuge
• Vortex

Pilot Experiment Procedure

Set up yeast

1. Collect 1 ml of yeast solution (approximately 1 million cells)
2. Centrifuge for 3 minutes at 1000x gravity
3. Remove supernatant
4. Add 1 ml of PBS buffer to yeast pellet
5. Vortex for 30 seconds

Make Fermentation Solution

1. Mix the following ingredients for each solution *See table 1
2. Vortex solution for 30 seconds
3. Store in 30°C incubator
4. Sample solution (for ethanol production) in 24 and 48 hours.

Table 1

	Solution 1	Solution 2	Solution 3	Solution 4
Yeast	1ml	1ml	1ml	1ml
Growth solution	8ml	8ml	8ml	8ml
40% Sucrose Solution	1ml	0.5ml	0.25ml	0.125ml
H20	0ml	0.5ml	0.75ml	0.875ml
Total Volume	10ml	10ml	10ml	10ml
Total sucrose concentration in solution	4%	2%	1%	0.5%

Results

We found through qualitative analysis that the 4% total sucrose in solution produced the most CO2 after 48 hours.

Knockout Procedure

Set up yeast

1. Collect 1 ml of yeast solution (approximately 1 million cells)
2. Centrifuge for 3 minutes at 1000x gravity
3. Remove supernatant
4. Add 1 ml of PBS buffer to yeast pellet
5. Vortex for 30 seconds

Make Fermentation Solution

1. Follow solution 1 in table 1 for each gene
2. Vortex solution for 30 seconds
3. Store in 30°C incubator for 48 hours

Prepare for HPLC

1. Take 1ml aliquot from fermentation
2. Centrifuge for 2 minutes at 15000x gravity
3. add 500ul of supernatant and 500ul of DI water to HPLC test tubes
4. Set up HPLC machine and run using standards that test for ethanol, glucose, and fructose.

Results of knockout experiment

All measurements are based off of 500ul of solution

Table 2

Gene	Glucose	Fructose	Ethanol
Standard solution	2.0000	0.2000	2.0000
YDl109C	0.3800	0.3933	0.3430
YGL140C	0.2212	0.2685	0.1867
YOR111W	0.3332	0.3598	0.1343
YHL029C	0.3870	0.2368	0.1151
YDR307W	0.4366	0.2487	0.0606
YNL058C	0.2710	0.3056	0.1577
YCL049C	0.4078	0.3052	0.1969
YGR079W	0.4042	0.1589	0.0080
YBL113C	0.3498	0.2012	0.1434
BY4735	0.3171	0.3084	0.3541