20.109(S23):M2D7

Introduction

Cell viability assay
The BacTiter-Glo Microbial Cell Viability Assay is a method for quantifying the number of viable cells based on measuring the amount of ATP present. ATP is a proxy for the presence of metabolically active (alive) cells. In this assay, the cells are lysed and ATP is released from the active cells. In a reaction catalyzed by a propriety luciferase enzyme, luciferin, ATP, and oxygen result in oxyluciferin, AMP, PP_i, carbon dioxide, and light. The light product is then measured using a luminometer.

Protocols

Part 1: Perform metal tolerance experiment

1. Obtain your Fet4_mutant culture from the front bench.
   • Untransformed W303α and W303α transformed with Fet4 will be tested in parallel by the teaching faculty.
2. Gently tritruate with a 1ml pipette to create a homogeneous suspension.
3. Obtain cuvettes from the front bench to measure the OD₆₀₀ of each culture prior to beginning the experiment.
4. Add 1ml of each cell suspension to individual cuvettes and read on the spectrophotometer.
   • Use 1ml SD-G for a blank.
     

     

     BacTiter-Glo assay map.
5. Record these numbers in your notebook.
6. Using SD-G media as a diluent, dilute each of your cultures to OD₆₀₀ ~ 1.0 and a final volume of 8ml. This does not have to be exact, but the cultures should have a similar OD₆₀₀ before you begin the experiment.
7. Add CdCl₂ for a final concentration of 100μM in each culture.
8. Incubate your labeled tubes shaking at 30°C for 2.5 hours to allow uptake.
   • During this incubation time, complete Part 2 and 3 of the wiki.
9. Following incubation, take an additional OD₆₀₀ reading to account for any changes in culture density, and allow normalization of data across groups.
   • Record this in your notebook.
10. Add 100μL of cell suspension to the appropriate wells as shown in the plate map to the right (Fet4_mutant cells -/+ cadmium will be assayed in triplicate).
11. Add 100μL BacTiter-Glo reagent to each well containing cell suspension and mix.
12. Incubate plate on orbital shaker, protected from light, for 5 minutes.
13. Read the plate on a luminometer plate reader.

In your laboratory notebook, complete the following:

• What

Part 2: Review collection of ICP-OES data

In your laboratory notebook, complete the following:

• What

Part 3: Analyze ICP-OES data

In your laboratory notebook, complete the following:

• What

Reagents list

• BacTiter-Glo Microbial Cell Viability Assay solution (Promega)
• Synthetic dropout - galactose (SD-G) media: 0.17% yeast nitrogen base without amino acid and ammonium sulfate (BD Bacto), 0.5% ammonium sulfate (Sigma), 0.192 % amino acid mix lacking uracil (Sigma), 2% raffinose (Sigma), 2% galactose (Sigma), 0.1% adenine hemisulfate (Sigma)
• Cadmium chloride (Sigma), stock concentration= 100mM

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