Difference between revisions of "20.109(S22):M2D6"

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(Reagents list)
(Part 3: Analyze fermentation product yield data)
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Because you will complete stastical analyses on these data, do not average the replicates until after samples are corrected for dilutions and normalized for cell density!
 
Because you will complete stastical analyses on these data, do not average the replicates until after samples are corrected for dilutions and normalized for cell density!
  
#Review the data in the Excel spreadsheet.
+
#Label the data so it is clear which conditions are shown in each column and row of your Excel spreadsheet.
#*Each tab contains the OD<sub>600</sub> and A<sub>570</sub> measurements acquired as described above.
+
#*The data are organized according to the plate map / steps above.
+
#Label the data so it is clear which conditions are shown in each column and row of the Excel spreadsheet.
+
 
#First, correct for the background ‘noise’ in your data by subtracting the averaged A<sub>570</sub> value of your 0 nmole/&mu;L samples from the A<sub>570</sub> values of all other samples (standards and experimentals).
 
#First, correct for the background ‘noise’ in your data by subtracting the averaged A<sub>570</sub> value of your 0 nmole/&mu;L samples from the A<sub>570</sub> values of all other samples (standards and experimentals).
 
#Average the background-corrected A<sub>570</sub> values for the replicates of the standard curve samples.
 
#Average the background-corrected A<sub>570</sub> values for the replicates of the standard curve samples.
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#*Divide the concentration of ethanol by the appropriate OD<sub>600</sub> value.
 
#*Divide the concentration of ethanol by the appropriate OD<sub>600</sub> value.
 
#Lastly, average the normalized values for each experimental replicate.
 
#Lastly, average the normalized values for each experimental replicate.
#Plot the results for each data set and include measures of variability (i.e. confidence intervals) and significance (i.e. p-values).
+
#Plot the results for each condition and include measures of variability (i.e. confidence intervals) and significance (i.e. p-values).
 +
#Upload your spreadsheet to the last column in the 'sgRNA_target sequences' table on the [[20.109(S22):Class_data#sgRNA_target_sequences| Class data tab]].
  
 
<font color =  #4a9152 >'''In your laboratory notebook,'''</font color> complete the following:
 
<font color =  #4a9152 >'''In your laboratory notebook,'''</font color> complete the following:

Revision as of 13:13, 25 January 2022

20.109(S22): Laboratory Fundamentals of Biological Engineering

Sp17 20.109 M1D7 chemical structure features.png

Spring 2022 schedule        FYI        Assignments        Homework        Class data        Communication        Accessibility

       M1: Drug discovery        M2: Metabolic engineering        M3: Project design       


Introduction

Protocols

Part 1: Measure cell density of cultures

Before ethanol yield is measured, it is important that to measure the optical density (OD), or density. As a reminder, 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 / acetate to the number of cells in each culture. This will eliminate biases due to growth rate and metabolic pathway. As an example, cells grown aerobically grow faster than cells grown anaerobically.

  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 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 calculations for product yield.
  5. Use the spectrophotometer to measure the OD600 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.

Part 2: Measure fermentation product yield

To ensure the steps included below are clear, please watch the video tutorial linked here: [Ethanol assay]. The steps are similar in both the ethanol and acetate assays!

Measure ethanol yield

Prepare supernatants from overnight cultures

  1. Transfer the cultures from your aerobic tubes to screw-cap tubes.
    • Be sure to label your tubes!
  2. Pellet the bacterial cells in your cultures by centrifugation using the large centrifuge.
    • 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 screw-cap tube into a fresh microcentrifuge tube.
    • Be sure to label your tubes!
  4. Label 8 additional microcentrifuge tubes and generate 1:15 dilutions of each of your samples in a final volume of 300 μL.
    • Use fresh LB as the diluent.

Prepare samples for standard curve

  1. Prepare a 1 nmole/μL stock ethanol solution:
    • In an microcentrifuge 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 microcentrifuge 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 microcentrifuge 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 5 microcentrifuge tubes as follows: 2 nmole, 4 nmole, 6 nmole, 8 nmole, and 10 nmole. (These will be the amounts in 50 μL samples, as will make sense in a few minutes.) Use the following steps to prepare samples for your standard curve that contain the specified amounts of ethanol.
    • In the 2 nmole tube, combine 144 μL of the Ethanol Assay Buffer with 6 μL of the 1 nmole/μL stock ethanol solution you generated in Step #1.
    • In the 4 nmole tube, combine 138 μL of the Ethanol Assay Buffer with 12 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 three times 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).

Complete ethanol assay

Plate map for ethanol assay setup.
  1. You will prepare your assay plate according to the map above.
    • Add 50 μL of the Ethanol Assay Buffer to wells A1 and B1. This is the 0 nmole/well sample for your standard curve.
    • Add 50 μL of each of the remaining standard curve samples to the appropriate wells.
    • Add 50 μL of your experimental samples according to the following order:
      • To C1, D1 and E1 add MG1655 +O2 -aTc
      • To C2, D2 and E2 add MG1655 -O2 -aTc
      • To C3, D3 and E3 add MG1655 +O2 +aTc
      • To C4, D4 and E4 add MG1655 -O2 +aTc
      • To C5, D5 and E5 add MG1655 +CRISPRi +O2 -aTc
      • To C6, D6 and E6 add MG1655 +CRISPRi -O2 -aTc
      • To C7, D7 and E7 add MG1655 +CRISPRi +O2 +aTc
      • To C8, D8 and E8 add MG1655 +CRISPRi -O2 +aTc
  2. Calculate the amount of Reaction Mix you will need for your assay using the following information:
    • To account for pipetting error, you will add an additional 20% of each component to the mixture.
    • You will add 50 μL of Reaction Mix to every well (supernatants and standard curve samples).
    • 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 a fresh, labeled conical tube.
  3. 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.
  4. Cover your 96-well plate tightly with aluminum foil and incubate at room temperature for 60 min.
    • Because the laboratory spectrophotometer is only able to measure a single sample in a cuvette, we will use a plate reader spectrophotometer to measure the A570 values for your assay.

Measure acetate yield

Prepare cell lystates from overnight cultures

  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 1:50 dilutions of each of your samples in a final volume of 200 μL.
    • Use cold Acetate Assay Buffer as the diluent.

Prepare samples for standard curve

  1. Prepare a 1 nmole/μL Acetate Stock solution:
    • In an eppendorf tube combine 990 μL of dH2O with 10 μL of the 100 mM acetate standard
  2. Label 6 eppendorf tubes as follows: 2 nmole, 4 nmole, 6 nmole, 8 nmole, 10 nmole and 12nmole. Use the following steps to prepare samples for your standard curve that contain the specified amounts of acetate.
    • In the 2 nmole tube, combine 144 μL of the Acetate Assay Buffer with 6 μL of the 1 nmole/μL stock Acetate solution you generated in Step #1.
    • In the 4 nmole tube, combine 138 μL of the Acetate Assay Buffer with 12 μL of the 1 nmole/μL stock Acetate solution you generated in Step #1.
    • Prepare the 6 nmole, 8 nmole, 10 nmole, and 12nmole tubes.
    • Note: the amount of Acetate in each tube is three times 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 Acetate in each well).

Complete acetate assay

Plate map for Acetate assay setup.
  1. You will prepare your assay plate according to the map above.
    • Add 50 μL of the Acetate Assay Buffer to wells A1 and B1. This is the 0 nmole/well sample for your standard curve.
    • Add 50 μL of each of the remaining standard curve samples to the appropriate wells.
    • Add 50 μL of your experimental samples according to the following order:
      • To C1, D1 and E1 add MG1655 +O2 -aTc
      • To C2, D2 and E2 add MG1655 -O2 -aTc
      • To C3, D3 and E3 add MG1655 +O2 +aTc
      • To C4, D4 and E4 add MG1655 -O2 +aTc
      • To C5, D5 and E5 add MG1655 +CRISPRi +O2 -aTc
      • To C6, D6 and E6 add MG1655 +CRISPRi -O2 -aTc
      • To C7, D7 and E7 add MG1655 +CRISPRi +O2 +aTc
      • To C8, D8 and E8 add MG1655 +CRISPRi -O2 +aTc
  2. Calculate the amount of Reaction Mix you will need for your assay using the following information:
    • To account for pipetting error, you will add an additional 20% of each component to the mixture.
    • You will add 50 μL of Reaction Mix to every well (cell supernatant and standard curve samples).
    • The Reaction Mix consists of 42 μL of the Acetate Assay Buffer, 2 μL of Acetate Substrate Mix, 2 μL of Acetate Enzyme Mix, 2 μL of Probe, and 2 μL of ATP per 50 μL.
    • Confirm your calculations with the teaching faculty before you proceed.
    • Combine the appropriate amount of cold Acetate Assay Buffer, Acetate Substrate Mix, Acetate Enzyme Mix, Probe, and ATP in a fresh, labeled eppendorf tube to make your master reaction mix for all wells.
  3. Add 50 μL of your Reaction Mix to each well in your 96-well plate that contains either cell supernatant or standard curve samples.
    • After each addition, use the pipet to mix the contents of the well.
    • Be sure to change pipet tips between additions.
  4. Cover your 96-well plate tightly with aluminum foil and incubate at room temperature for 40 min.
  5. 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 to measure the A450 values for your assay.

Part 3: Analyze fermentation product yield data

  1. Once you obtain your raw data, please use this excel template and post it to the [class data page] to share with your classmates.

Today you will analyze the ethanol yield data that was generated using the sgRNA_target sequences you aligned in the previous laboratory session. The data you will use in this exercise is provided in an Excel spreadsheet with each dataset as a separate tab (linked here). Because a total of 12 experiments will be analyzed, it may be helpful to prepare a template in the Excel spreadsheet that will automatically calculate the ethanol yield when populated with the data for each experiment. As before, feel free to divide the workload between laboratory partners.

Because you will complete stastical analyses on these data, do not average the replicates until after samples are corrected for dilutions and normalized for cell density!

  1. Label the data so it is clear which conditions are shown in each column and row of your Excel spreadsheet.
  2. First, correct for the background ‘noise’ in your data by subtracting the averaged A570 value of your 0 nmole/μL samples from the A570 values of all other samples (standards and experimentals).
  3. Average the background-corrected A570 values for the replicates of the standard curve samples.
  4. Plot the A570 values for your standard curve samples.
    • Provide the EtOH concentration on the y-axis and the A570 on the x-axis.
    • Include the R2 value and the equation of the best-fit line on your graph.
  5. Use the equation of the best-fit line to calculate the amount of ethanol (nmol) in the experimental samples.
    • Remember that a dilution of the supernatant was used in the assay!
    • For each dataset, use the standard curve curve samples that were prepared on the same plate as the experimental samples.
  6. Next, calculate the concentration of product using the following equation: Sa / Sv = C
    • Sa = amount of fermentation product in unknown sample (nmole) from standard curve
    • Sv = sample volume (μL) added to well
    • C = concentration of ethanol in sample
  7. Then, normalize the concentration of ethanol calculated to the OD600 of the cell cultures to account for difference in cell number.
    • Divide the concentration of ethanol by the appropriate OD600 value.
  8. Lastly, average the normalized values for each experimental replicate.
  9. Plot the results for each condition and include measures of variability (i.e. confidence intervals) and significance (i.e. p-values).
  10. Upload your spreadsheet to the last column in the 'sgRNA_target sequences' table on the Class data tab.

In your laboratory notebook, complete the following:

  • Attach the Excel spreadsheet with the ethanol yield data analyzed.
  • For each dataset, are the results expected? Why or why not?
  • Are you confident in the results? Why or why not?
  • How do these results compare to the speculations you made regarding which sgRNA_target sequences might be better at increasing ethanol yield?

Reagents list

  • Luria-Bertani (LB) broth: 1% tryptone, 0.5% yeast extract, and 1% NaCl
  • Ethanol Assay Kit (from Sigma)
    • ethanol assay buffer
    • ethanol probe
    • ethanol enzyme
  • Acetate Assay Kit (from Abcam)
    • acetate assay buffer
    • acetate probe
    • acetate substrate
    • adenosine triphosphate (ATP)
    • acetate enzyme

Navigation links

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