Difference between revisions of "20.109(F16)::Measure fermentation products (Day8)"

Introduction

Protocols

Before you prepare your samples to quantify the fermentation product yield, it is important that you measure the optical density (OD) of your cultures. This number will be used to normalize your data, which allows you to more accurately compare your samples. As a reminder, the OD is a measure of culture density and is based on the absorbance of light at 600 nm (A₆₀₀). In this method of analysis, the higher the OD is the more dense the culture.

1. Obtain an aliquot of fresh LB media and 9 plastic cuvettes from the front laboratory bench.
2. Pipet 1000 μL of LB into 1 cuvette. This will serve as the blank needed to calibrate the spectrophotometer.
3. Pipet 900 μL of LB into each of the 8 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 A₆₀₀ 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 calculations for product yield.
5. Use the spectrophotometer to measure the A₆₀₀ values according to the instructions from the Orientation exercise.
   • This step will be a bottleneck. Please be courteous to your laboratory mates and do not go to the spectrophotometer until your samples are fully prepared and ready to be measured.

Measuring ethanol yield

Prepare supernatant

1. Transfer the cultures from your aerobic tubes to fresh 15-mL conical tubes.
   • Be sure to label your tubes!
2. Pellet the bacterial cells in your cultures by centrifugation using the large centrifuge on the teaching faculty laboratory bench.
   • Alert the teaching faculty when you are ready for centrifugation.
   • Centrifuge your samples at 3000 rpm for 10 min.
   • If the media is still ‘cloudy’ repeat the centrifugation step.
3. Pipet 1000 μL of the supernatant from each 15-mL conical tube into a fresh 1.5-mL eppendorf tube.
   • Be sure to label your tubes!
4. Label 8 additional eppendorf tubes and generate 1:10 dilutions of each of your samples in a final volume of 1000 μL.
   • Use fresh LB as the diluent.

Prepare standard curve samples
The Ethanol Assay Buffer and 17.15 N Ethanol Standard are at the front laboratory bench.

1. Prepare a 1 nmole/μL stock ethanol solution:
   • In an eppendorf tube labeled A, combine 808.7 μL of the Ethanol Assay Buffer with 50 μL of the 17.15 N Ethanol Standard to generate a 1 μmole/μL solution.
   • In an eppendorf tube labeled B, combine 990 μL of Ethanol Assay Buffer with 10 μL of solution A (from tube A) to generate a 10 nmole/μL solution.
   • In an eppendorf tube labeled C, combine 900 μL of Ethanol Assay Buffer with 100 μL of solution B (from tube B) to generate a 1 nmole/μL stock ethanol solution.
2. Label 4 eppendorf tubes as follows: 2 nmole, 4 nmole, 6 nmole, 8 nmole, and 10 nmole. Use the following steps to prepare samples for your standard curve that contain the specified amounts of ethanol.
   • In the 2 nmole tube, combine 96 μL of the Ethanol Assay Buffer with 4 μL of the 1 nmole/μL stock ethanol solution you generated in Step #1.
   • In the 4 nmole tube, combine 92 μL of the Ethanol Assay Buffer with 8 μL of the 1 nmole/μL stock ethanol solution you generated in Step #1.
   • Prepare the 6 nmole, 8 nmole, and 10 nmole tubes.
   • Note: the amount of ethanol in each tube is twice as much as specified by the label because you are preparing enough for duplicate samples (ie the contents of each tube will be divided between two wells in your assay resulting in the appropriate amount of ethanol in each well).

Prepare samples for assay

1. Draw plate map!!

1. Calculate the amount of Reaction Mix you will need for your assay using the following information:
   • You will add 50 μL of Reaction Mix to every well (standards and experimentals).
   • The Reaction Mix consists of 46 μL of Ethanol Assay Buffer, 2 μL of Ethanol Probe, and 2 μL of Ethanol Enzyme Mix per 50 μL.
   • Confirm your calculations with the teaching faculty before you proceed.
   • Combine the appropriate amount of Ethanol Assay Buffer, Ethanol Probe, and Ethanol Enzyme Mix in fresh, labeled eppendorf tube.
2. Add 50 μL of your Reaction Mix to each well in your 96-well plate that contains either standard or experimental samples.
   • After each addition, use the pipet to mix the contents of the well.
   • Be sure to change pipet tips between additions.
3. Cover your 96-well plate tightly with aluminum foil and incubate at room temperature for 60 min.
4. Alert the teaching faculty when your samples are ready for the spectrophotometer.
   • Because the laboratory spectrophotometer is only able to measure a single sample in a cuvette, we will use a plate reader spectrophotometer in the BMC to measure the A₅₇₀ values for your assay.

Data analysis

1. Prepare a spreadsheet with the spectrophotometer values obtained for your assay.
   • Average the technical replicates in your data set.
2. Correct for the background ‘noise’ in your data by subtracting the averaged value of your 0 nmole/μL samples from the averaged values of all other samples.
3. Plot the corrected values for your standard curve samples with the ethanol concentration on the y-axis and the A₅₇₀ on the x-axis.
   • Include the R-squared value and the equation of the best-fit line on your graph.
   • Attach the graph to your Benchling laboratory notebook entry.
4. Use the equation of the best-fit line to calculate the amount of ethanol in your experimental samples.
   • Remember that you used a dilution of the supernatant in your assay!
5. If you choose to report your data as concentration of ethanol produced, use the following equation: S_a / S_v = C
   • S_a = amount of ethanol in unknown sample (nmole) from standard curve
   • S_v = sample volume (μL) added to well
   • C = concentration of ethanol in sample
   • The units of this calculation will be nmole/μL. For units of ng/μL use the molecular weight value of ethanol, which is 46.07 g/mole.
6. Lastly, normalize your data to the OD of your initial culture to account for difference in cell number.
   • Divide the amount or concentration of ethanol produced by the OD value measured.

Measuring lactate yield

Reagents

• Ethanol Assay Kit (Sigma-Adrich)

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Next day: Module 3 starts!