View source for 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====
# Collect 1 ml of yeast solution (approximately 1 million cells)
# Centrifuge for 3 minutes at 1000x gravity
# Remove supernatant
# Add 1 ml of PBS buffer to yeast pellet
# Vortex for 30 seconds

====Make Fermentation Solution====
# Mix the following ingredients for each solution *See table 1
# Vortex solution for 30 seconds
# Store in 30°C incubator
# Sample solution (for ethanol production) in 24 and 48 hours.

===Table 1===

{| border="1" cellspacing="4" cellpadding="2"
|
|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====
# Collect 1 ml of yeast solution (approximately 1 million cells)  
# Centrifuge for 3 minutes at 1000x gravity
# Remove supernatant
# Add 1 ml of PBS buffer to yeast pellet
# Vortex for 30 seconds

====Make Fermentation Solution====
# Follow solution 1 in table 1 for each gene
# Vortex solution for 30 seconds
# Store in 30°C incubator for 48 hours

====Prepare for HPLC====
# Take 1ml aliquot from fermentation
# Centrifuge for 2 minutes at 15000x gravity
# add 500ul of supernatant and 500ul of DI water to HPLC test tubes
# 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===
{| border="1" cellspacing="4" cellpadding="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
|}