BE FPOP(Su24):Measuring ethanol yield

Introduction

To measure the effect the sgRNA_targets on gene expression using the CRISPRi system, you will test the E. coli cultures you inoculated on Day 1 with commercially available kits that quantify the amount of ethanol in the supernatent. Though the reagents for the kit is proprietary, we do know that it utilizes enzymatic reactions to produce a colorimetric readout. In this, the darker the purple color of the sample the more ethanol is present.

Protocols

Measure cell density of cultures

Before you prepare your cultures for the ethanol yield assay it is important to measure the optical density (OD), or density. The OD is a measure of culture density and is based on the deflection of light at 600 nm (OD₆₀₀). In this method of analysis, the higher the OD is the more dense the culture. Because there are variables inherent in the experimental setup it is important to normalize the amount of ethanol produced to the number of cells in each culture. This will eliminate biases due to growth rate differences that may occur between culture tubes.

1. Obtain an aliquot of fresh LB media and six plastic cuvettes from the front laboratory bench.
2. Pipet 1000 μL of LB into one cuvette.
   • This will serve as the blank needed to calibrate the spectrophotometer.
3. Pipet 900 μL of LB into each of the five remaining cuvettes.
   • It may be helpful to label your cuvettes with a simple designation that corresponds to your samples. Be sure that your labels are not in the light path as this will obscure your OD₆₀₀ readings.
4. Transfer 100 μL of each culture into the appropriate plastic cuvette.
   • Note: this is a 1:10 dilution of your sample that should be accounted for in your later calculations for product yield!
5. Use the spectrophotometer to measure the OD₆₀₀ values.
6. In your laboratory notebook, complete the following:
   • Record the OD₆₀₀ readings for your samples.
   • Calculate the actual OD₆₀₀ values for your samples to account for the 1:10 dilution.

Prepare cultures for ethanol yield assay

For this step, you will remove the bacterial cells from your cultures and use the supernatent to measure the ethanol yield from each culture.

1. Transfer 1 mL from each of your cultures into labeled microcentrifuge tubes.
   • Centrifuge your samples at max speed for 1 min.
2. Pipet 20 μL of the supernatent from each sample into a clean, labeled microcentrifuge tube.
3. In your laboratory notebook, calculate the amount of Working Reagent you will need for your experiment.
   • For each reaction you will need the following:
     • 80 μL of Assay Buffer
     • 1 μL of Enzyme A
     • 1 μL of Enzyme B
     • 2.5 μL of NAD Solution
     • 14 μL of MTT Solution
   • For each of your samples you will prepare duplicate reactions.
   • Check your math with the FPOP team before continuing to Step #5.
4. In a microcentrifuge tube, prepare the Working Reagent for your samples.
   • Use the vortexer to completely mix the solution.
5. Add 180 μL of Working Reagent into each of the tubes you prepared in Step #2.
   • Mix completely by pipetting the solution up and down.
   • Be sure to change pipet tips between tubes!
6. Close the tubes and incubate for 30 minutes at room temperature.
7. After the incubation, add 200 μL of Stop Reagent to each of the tubes.
   • Mix completely by pipetting the solution up and down.
   • Be sure to change pipet tips between tubes!
8. Transfer your reactions to the 96-well plate located on the front laboratory bench according to the plate map provided at the right.
   • Pipet 200 μL from each tube into two wells for duplicate samples.
   • Be sure to change pipet tips between tubes!
     
9. In your laboratory notebook, note the visual comparison of the 'purpleness' of your samples, which condition has the most ethanol? The least ethanol?
10. When all teams have transferred their samples to the 96-well plate, the A₅₆₅ values will be measured using a plate spectrophotometer.
11. In your laboratory notebook, record the A₅₆₅ readings for each of your samples.