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Eau That Smell

Compare two competing designs to optimize system performance

OBJECTIVES

By the conclusion of this laboratory investigation, you should be able to:

INTRODUCTION

For the 2006 iGEM competition, MIT students designed Eau d’coli, E. coli that smell like bananas when their population is in the stationary phase. They did this by inserting a device that contains a stationary phase sensitive promoter coupled to a banana smell device. The banana smell device contained a ribosome binding site (RBS), an open reading frame (ORF) that encodes the ATF1 enzyme and terminator sequences. The ATF1 enzyme converts isoamyl alcohol to isoamyl acetate, the molecule that gives bananas their characteristic smell.

In this investigation, you’ll try to generate the banana smell during the bacteria’s log phase of population growth. There are two ways (at least!) you could accomplish this. Both approaches use the original banana smell generator device (an RBS, the ATF1 gene and a transcriptional terminator). One approach couples the banana smell generator device to a new part, a log phase promoter. The other approach uses the stationary phase promoter that was used in the original MIT project but adds a genetic inverter between the stationary phase promoter and the banana smell generator device.

These variants have already been made for you and transformed into bacteria. In fact, you have been sent four strains of E. coli to test. Each contains a different device:

  • Sample 1-1. The original Eau d’coli device
  • Sample 1-2. The original Eau d’coli device but with an inverter added between the promoter and the RBS.
  • Sample 1-3. The banana smell generator coupled to the log phase promoter
  • Sample 1-4. A strain of E. coli that has no smell generating devices.

Your task will be to grow liquid cultures of these bacteria and measure the intensity of the banana smell as the population moves from lag phase through log phase and into stationary phase. The intensity of the banana smell can be compared to dilutions of banana extract. The population growth can be measured at each of these phases using a spectrophotometer or the McFarland Turbidity Standards.

Procedure

There are two versions of this lab. Protocol A is the shorter version, with only a measurement or two at each phase of growth. Protocol B is a longer version of this experiment, with measurements made throughout the entire population growth curve.

Lab version

DAY 1

Your teacher will inform you if you are performing this part of the protocol or if it has been done for you.

We will be receiving our bacterial strains with the plasmids already inserted. The strains may come in the form of a “stab” or “slant,” a test tube with a small amount of bacteria on a slanted media which will be innoculated into media on Day 2.

If you have LB+Amp petri dishes handy, you can streak the strains as described below. This is not necessary but can be useful if several teams are setting up overnight liquid cultures as described for Day 2.

PROCEDURE for streaking strains to petri dishes

Using a sterile toothpick or inoculating loop, gather a small amount of bacteria from the stab and transfer it to a petri dish containing Luria Broth (LB) agar plus ampicillin.

  1. Repeat with the remaining stab samples, streaking out each onto a different petri dish.
  2. Place these petri dishes media side up in a 37°C incubator overnight.

Watch how you streak out cells. You should wear gloves though!!

Streaking Cells

DAY 2

Your teacher will inform you if you are performing this part of the protocol or if it has been done for you.

If the liquid overnights have already been grown, you can proceed to Day 3.

PROCEDURE
  1. Using a sterile inoculating loop or toothpick or pipet tip, transfer a loopful of cells from the stab (or a bacterial colony from one of the petri dishes if you’ve streaked them out yesterday) to a 15ml sterile culture tube containing 3 ml of Luria Broth (LB) + ampicillin.
  2. Repeat for each strain you will inoculate.
  3. Place the culture tubes in the roller wheel in the incubator at 37°C overnight. Be sure to balance the tubes across from each other to minimize stress on the roller wheel. Alternatively, tubes can be placed on a platform shaker to grow the cells with aeration overnight.

Watch how you grow overnight cultures. You should wear gloves though!!

Grow Liquid Overnights

DAY 3

Your teacher will inform you if you are performing this part of the protocol or if it has been done for you.

If the bacterial cultures have already been grown to lag, log and stationary phase, you can proceed to Day 4.

PROCEDURE
  1. Prepare a stock growth solution with
    • 300 ml Luria broth
    • 300 μl Ampicillin (final concentration= 100 mg/liter)
    • 250 μl isoamyl alcohol
  2. Mix this stock growth solution, by swirling the bottle or vortexing gently.
  3. If you will be using a spectrophotemeter, set aside 2 ml of this mixture into a small sterile culture tube. This aliquot will serve as the blank for the spectrophotometer. Store this in the refrigerator.
  4. Move 75 ml of the broth solution to 125ml sterile erlenmeyer flask and add 2ml of bacteria from one of the overnight cultures, e.g. strain 1-1.
  5. Repeat the addition of 2ml of bacteria to 75 ml of broth in the erlenmeyer flasks for each of the overnight cultures.
  6. Label four 50 ml conical tubes: Label each tube T0 and indicate the bacterial strain (e.g. 1-1).
  7. Remove 25 mls from culture 1-1 and place in the conical tube and store in the refrigerator. This will be the lag phase sample you will read on Day 4.
  8. Repeat the previous step for each culture.
  9. Cover the flasks with foil and start them gently stirring on the stir plates for 5-7 hours. This is done at room temperature. Record the time each culture started spinning.
  10. Label four 50 ml conical tubes: Label each tube Tlog and indicate the bacterial strain (e.g. 1-1). Record the number of minutes since the culture started spinning.
  11. Remove 25 mls from culture 1-1 and place in the conical tube and store in the refrigerator. This will be the log phase sample you will read on Day 4.
  12. Repeat the previous step for each culture.
  13. Allow the remaining cultures to incubate overnight on the stir plates at room temperature. These will be the stationary phase samples you will read on Day 4.

Watch how you grow bacterial cultures. You should wear gloves though!!

Grow Bacterial Cultures

DAY 4

Your teacher will inform you if you are performing this part of the protocol or if it has been done for you.

PROCEDURE IF USING A SPECTROPHOTOMETER

  1. Remove 2 ml from each sample to read lag phase density of each. If you are testing all 4 samples you should now have 5 small test tubes, 4 with bacterial samples and one blank (the media you saved on Day 3).
  2. Prepare the spectrophotometer by setting it to OD600.
  3. Read the blank and adjust the % Absorbance to zero.
  4. Read the sample tubes and record the % Absorbance.
  5. Sniff the flask for any evidence of a banana smell, comparing the smell with the banana extract standards. Be sure to shake the standards and the cultures before sniffing. Record your data.
  6. Repeat the above steps with the log phase samples and then the stationary phase samples.
  7. Calculate the bacterial population: 1 OD600 unit = 1 x 109 bacteria.

 

PROCEDURE, IF NO SPECTROPHOTOMETER IS AVAILABLE

The turbidity of the bacterial populations can be estimated using the McFarland Turbidity Scale. This method uses suspensions of a 1% BaCl2 in 1% H2SO4 that are visually similar to suspensions of various populations of E. coli.

    1. Remove 2 ml from each sample to read lag phase density of each. If you are testing all 4 samples you should now have 4 small test tubes.
    2. Following your teacher’s instructions, obtain small clear test tubes containing the turbidity standards. The tubes should contain enough standard in each to fill the tube to a height of about 1 inch (2.5 cm) from the bottom. Make sure each tube is properly labeled with its turbidity standard number. If you are filling the tubes from stock bottles of the standards, use small tubes and place enough standard in each to fill the tube to a height of about 1 inch (2.5 cm) from the bottom.
    3. Place the standards in a test tube rack that allows you to view them from the side. Use small tubes and place enough standard in each to fill the tube to a height of about 1 inch (2.5 cm) from the bottom.
    4. On a blank index card or paper use a marker to draw two thick black lines. These lines should be within the height of the standards.
    5. Place the card with the lines behind the standards.
  1. To compare your bacterial cultures to the standards, you will need to place the bacterial sample in a test tube of the same size and equal volume as the standards. Be sure to label these sample tubes.
  2. Place the sample tube next to the standard tubes. You should move the sample to compare it to the standard tubes with the most similar turbidity. You can make this assessment more precise by looking for a standard that most similarly obscures the black lines on the background card.
  3. Use the table below to determine the comparable OD 600.
  4. 1 OD 600 unit equals approximately 1 x 109 cells.
  5. Sniff the flask for any evidence of a banana smell, comparing the smell with the banana extract standards. Be sure to shake the standards and the cultures before sniffing. Record your data.
  6. Repeat the above steps using the McFarland Standards with the log phase samples and then the stationary phase samples.

LAB REPORT

Your report on this investigation may include:

  1. Introduction
    • Provide a brief introduction describing the field of synthetic biology.
    • Briefly describe the purpose of the lab. What are we trying to do here?
    • Explain how the banana smell generator functions.
    • Why are we using optical density to measure the population?
    • Explain each phase of the bacterial population growth curve.
    • Presume that a reader of your lab report has not read the assignment.
  2. Methods
    • You do not have to rewrite the procedure.
    • Explain why you did each step of the protocol.
  3. Results
    • Present the data tables in clear format.
    • Draw population growth curves of the class mean data for each sample. Indicate on each curve when you could smell bananas.
  4. Discussion
    • Describe the results: Were we able to measure the population growth? Were we able to smell bananas? Did each device produce the same results? Did the genetic systems affect the growth curve of the bacteria? Explain your answers.
    • Analyze the data: Be sure to discuss how each part of the experiment adds to your conclusion.
    • Discuss errors and other reasons for data variability.
    • How confident are you in the results? Are you equally confident in both the growth data and the smell data? Explain.
    • Is using smell to measure the banana smell valid? Why or why not?
    • What methods did you use to try to increase your confidence in the results?
    • How might we try to change this system so that we can quantify the banana smell? Would we be better off using a different kind of signal? If so, what would you suggest?
    • If you could construct a different genetic system, what might you construct? What would you need to do?
  5. Citations and references
    • Be sure these are of good quality.
    • Embed citations.
    • Follow proper reference format.

DAY 1

Your teacher will inform you if you are performing this part of the protocol or if it has been done for you.

We will be receiving our bacterial strains with the plasmids already inserted. The strains may come in the form of a “stab” or “slant,” a test tube with a small amount of bacteria on a slanted media, in which case you will have to streak out the bacteria onto a petri dish to continue the experiment.

If the bacteria have arrived on petri dishes, you can proceed to Day 2.

PROCEDURE

Using a sterile toothpick or inoculating loop, gather a small amount of bacteria from the stab and transfer it to a petri dish containing Luria Broth (LB) agar plus ampicillin medium.

  1. Repeat with the remaining stab samples, streaking out each onto a different petri dish.
  2. Place these petri dishes media side up in a 37°C incubator overnight.

Watch how you streak out cells. You should wear gloves though!!

Streaking Cells

DAY 2

Your teacher will inform you if you are performing this part of the protocol or if it has been done for you.

If the liquid overnights have already been grown, you can proceed to Day 3.

PROCEDURE
  1. Using a sterile inoculating loop or toothpick or pipet tip, transfer a bacterial colony from one of the petri dishes to a large sterile culture tube containing 3 ml of Luria Broth (LB) + ampicillin.
  2. Repeat for each strain you will inoculate.
  3. Place the culture tubes in the roller wheel in the incubator at 37°C overnight. Be sure to balance the tubes across from each other to minimize stress on the roller wheel. Alternatively, tubes can be placed on a platform shaker to grow the cells with aeration overnight.

Watch how you grow overnight cultures. You should wear gloves though!!

Grow Liquid Overnights

DAY 3

PROCEDURE
  1. Prepare a stock growth solution with
    • 300 ml Luria broth
    • 300 μl Ampicillin (final concentration= 100 mg/liter)
    • 250 μl isoamyl alcohol
  2. Mix this stock growth solution, by swirling the bottle or vortexing gently.
  3. Set aside 2 ml of this mixture for each student group into a small sterile culture tube. This aliquot will serve as the blank for the spectrophotometer.
  4. Move 75 ml of the broth solution to 125ml sterile erlenmeyer flask and add 2ml of bacteria from one of the overnight cultures, e.g. strain 1-1.
  5. Repeat the addition of 2ml of bacteria to 75 ml of broth in the erlenmeyer flasks for each of the overnight cultures.
  6. Add a stir bar to each culture flask and place onto stir plates. Stir slowly. Cover the flasks with foil.
PROCEDURE USING A SPECTROPHOTOMETER
  1. Remove 2 ml from each sample to read the starting density of each. If you are testing all 4 samples you should now have 5 small test tubes, 4 with bacterial dilutions and one blank.
  2. Prepare the spectrophotometer by setting it to OD600.
  3. Note the time and take an “initial” density reading for the bacterial samples. Please note that your teacher may have carried out the preceding steps in advance of the lab. If that is the case, the teacher will tell you how much time has elapsed.
  4. After 20 minutes, move 1 or 2 ml from each sample to cuvettes. Note: the volume you use here will depend on the size of the cuvette appropriate for your spectrophotometer. Please follow the teacher’s instructions.
  5. Read the blank and adjust the % Absorbance to zero.
  6. Read the sample tubes and record the % Absorbance.
  7. Sniff the flask for any evidence of a banana smell, comparing the smell with the banana extract standards. Be sure to shake the standards and the cultures before sniffing. Record your data.
  8. At 20 minute intervals repeat steps 4-7.
  9. Between time points, you can calculate the bacterial population: 1 OD600 unit = 1 x 109 bacteria.
PROCEDURE IF NO SPECTROPHOTOMETER IS AVAILABLE

The turbidity of the bacterial populations can be estimated using the McFarland Turbidity Scale. This method uses suspensions of a 1% BaCl2 in 1% H2SO4 that are visually similar to suspensions of various populations of E. coli.

  1. Following your teacher’s instructions, obtain small clear test tubes containing the turbidity standards. The turbidity standard solution should fill the tube to a height of about 1 inch (2.5 cm) from the bottom.
  2. Place the samples in a test tube rack that allows you to view them from the side.
  3. On a blank index card or paper use a marker to draw two thick black lines. These lines should be within the height of the standards.
  4. Place the card with the lines behind the standards.
MEASURING POPULATION GROWTH AND BANANA SMELL:
  1. Remove 2 ml from each sample to read the starting density of each. If you are testing all 4 samples you should now have 4 small test tubes, one for each strain. Replace the foil and keep each flask spinning slowly.
  2. Write down the time for your first time point in your data table and take an “initial” density reading for the bacterial samples. This will be your T = 0 unless your teacher or other students have done this inital reading for your already. If that’s the case, then you should find out how much time has elapsed since T = 0 and write that down in your data table. To take this reading, place the tube with your bacterial sample next to the tubes with the McFarland Standards. Compare the bacterial sample to the standard tubes and identify which standard has the most similar turbidity. Try to decide which standard most similarly obscures the black lines on the background card.
  3. Use the table below to determine the comparable OD 600. 1 OD 600 unit equals approximately 1 x 109 cells.
  4. After 20 minutes repeat the sampling steps above, but record the time as T=0 + 20.
  5. Sniff the flask for any evidence of a banana smell, comparing the smell with the banana extract standards. Be sure to shake the standards and the cultures before sniffing. Record your data.
  6. At 20 minute intervals repeat sampling steps. Record time as T=0 + the number of minutes since T=0.
  7. Your teacher will inform you of the length of this test and may provide you with data from other classes.

LAB REPORT

Your report on this investigation may include:

  1. Introduction
    • Provide a brief introduction describing the field of synthetic biology.
    • Briefly describe the purpose of the lab. What are we trying to do here?
    • Explain how the banana smell generator functions.
    • Why are we using optical density to measure the population?
    • Explain each phase of the bacterial population growth curve.
    • Presume that a reader of your lab report has not read the assignment.
  2. Methods
    • You do not have to rewrite the procedure.
    • Explain why you did each step of the protocol.
  3. Results
    • Present the data tables in clear format.
    • Draw population growth curves of the class mean data for each sample. Indicate on each curve when you could smell bananas.
  4. Discussion
    • Describe the results: Were we able to measure the population growth? Were we able to smell bananas? Did each device produce the same results? Did the genetic systems affect the growth curve of the bacteria? Explain your answers.
    • Analyze the data: Be sure to discuss how each part of the experiment adds to your conclusion.
    • Discuss errors and other reasons for data variability.
    • How confident are you in the results? Are you equally confident in both the growth data and the smell data? Explain.
    • Is using smell to measure the banana smell valid? Why or why not?
    • What did methods did you use to try to increase your confidence in the results?
    • How might we try to change this system so that we can quantify the banana smell? Would we be better off using a different kind of signal? If so, what would you suggest?
    • If you could construct a different genetic system, what might you construct? What would you need to do?
  5. Citations and references
    • Be sure these are of good quality.
    • Embed citations.
    • Follow proper reference format.

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