UW-Stout/pH FA21

Introduction

We will be stressing our yeast by exposing it to different pH and measuring its sensitivity based on its survival rate. In this experiment, we will be using 7.0, 6,5,4,3,2.6, 2.4,2.2 pH of as a control for the first 12 to 15 hours on wild type.

Materials

• Micropipettor
  • You will need the P20 (2-20µl)
• Micropipettor tips in blue, green and red boxes.
• Tranformed yeast
• Distilled Water
• 12 wells
• Citric acid
• Disodium phosphate
• 15mL tube
• Deionized water
• powder
• Filter
• incubator plate
• syringe
• Scale
• Corning COSTAR 96-well clear flat-bottom assay plate
• Falcon round-bottom polypropylene tubes
• YPD media, both broth and 2% agar plates
• 96 well plate
• Micro pipette tips
• 6 PCR tubes containing wild yeast and 5 knockout strains
• Sterile Water

Procedure

• Preparing Buffer stock solutions
• Read previous studies to determine what buffer would be suitable
• Determined that citrate/phosphate buffer would be most suitable for a buffer in this experiment
• preformed calculations to determine the amount of citric acid and disodium phosphate needed to make 1M stock solutions of both from those stock solutions, we can now make buffers of varying pH to torture our yeast in.

Sterilization

• mix powder and the water
• Use sterile syringe and a filter to pour the liquid into the syringe.
  • Use a clean tube for the liquid in the syringe to filter through in.
• sterilize distilled water

Three days before the experiment, streak knockout strains onto YPD plates. The morning before the experiment, pick a colony from each strain into 5 ml YPD broth in a disposable test-tube. Incubate on the roller drum at 30°C for 8-10 hours. The evening before the experiment, transfer 20 ul of each strain into 5 ml of fresh YPD broth. Incubate on a roller drum at 30°C overnight. The morning of the experiment, dilute each culture to an OD600 of 0.1-0.2 in YPD broth. Return to the drum roller until ready to test.

Preparing Buffer stock solutions

• Retrieve your citric acid and disodium phosphate
• Determine the amount of each needed to make 10mL of .10M stock solutions of disodium phosphate and citric acid.
  • From those stock solutions, buffer solutions can be made
• Thoroughly mix solutions to ensure all solids are dissolved

Equipment

• Incubator set to 30°C.
• New Brunswick TC-7 Tissue Culture Roller Drum
• Molecular Devices SpectraMax Plus 384 Microplate Reader

Sterilization

• Acquire 3-15mL syringes, 3-micron filters, and 3 new 15mL tubes.
• Attach micron filter to tip of syringe and remove plunger
• Pour first stock solution into syringe
• While holding syringe over UNUSED 15mL tube, insert plunger and push your solution through filter into tube, seal and label
• Repeat with second stock solution and DI water

Making buffer

pH 7

• Using a pipettor, pipet 329.4 uL .10M disodium phosphate into microcentrifuge tube
• Add 35.3 uL citric acid into same tube
• Add 635.3 uL sterile water into tube
• seal tube, vortex, and label pH 7

pH 6

• pipet 252.6 uL disodium phosphate into microcentrifuge tube
• Add 73.7 uL citric acid into same tube
• Add 673.7 uL sterile water into tube
• seal tube, vortex, and label pH 6

pH 5

• pipet 206 uL disodium phosphate into microcentrifuge tube
• Add 97 uL citric acid into same tube
• Add 697 uL sterile water into tube
• seal tube, vortex, and label pH 5

pH 4

• pipet 154.2 uL disodium phosphate into microcentrifuge tube
• Add 122.9 uL citric acid into same tube
• Add 722.9 uL sterile water into tube
• seal tube, vortex, and label pH 4

pH 3

• pipet 82.2 uL disodium phosphate into microcentrifuge tube
• Add 158.9 uL citric acid into same tube
• Add 758.9 uL sterile water into tube
• seal tube, vortex, and label pH 3

pH 2.6

• pipet 43.6 uL disodium phosphate into microcentrifuge tube
• Add 178.2 uL citric acid into same tube
• Add 778.2 uL sterile water into tube
• seal tube, vortex, and label pH 2.6

pH 2.4

• pipet 24.8 uL disodium phosphate into microcentrifuge tube
• Add 187.6 uL citric acid into same tube
• Add 787.6 uL sterile water into tube
• seal tube, vortex, and label pH 2.4

pH 2.2

• pipet 8 uL disodium phosphate into microcentrifuge tube
• Add 196 uL citric acid into same tube
• Add 796 uL sterile water into tube
• seal tube, vortex, and label pH 2.2

Addition of Buffer to Yeast

• Vortex Yeast cultures
• Add 50 uL yeast cells in each well for each pH level
• Add 50 uL of DI water to first well as a control
  • add 50uL pH buffe to each subsequent well descending in pH level

Setting up the plate Reader

• Temperature: at 30 degrees Celsius
• Mode: Kinetic
• Read: 600 n
• Intervals: 5 minutes
• Total run time: 24 hours
• Shake before read: 30 seconds
• Transfer the assay plate to the reader and run for 24 hours

RESULTS: GROWTH CURVE FOR CALIBRATION EXPERIMENT

Notes

From our calibration experiment, better known as our pilot experiment, we used all of our pH in order to see how much stress the wild type (BY4735) can take. Starting with DI water and ending with the pH of 2.2, we saw that none of the pH levels killed off the yeast. However, we noticed that pH of 2.6 provided a desirable amount of stress without stunting growth significantly.

Final Protocol

• REPEAT PILOT PROTOCOL USING ONLY pH 2.6
• Use the Wild Type strand, and the knockout strains
• repeat "preparing plate reader"
• Annotate graph curve