BioBuilding: Synthetic Biology for Teachers: Lab 1--Protocol B (expanded)

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Contents

Lab 1: Eau that smell--Protocol B

Image:Copy of BBa J45200.png

Teacher Considerations

This lab provides a valuable opportunity to teach microbiology techniques, population growth dynamics, molecular genetics and basic synthetic biology concepts in a meaningful, real world way. As can be seen in the discussion questions for the lab report, the analysis of the lab will provide the students with a chance to do meaningful error analysis and examine the difference between quantitative results and qualitative results.

Protocol B--Growth curve data collection: This version provides a greater emphasis on collection of growth curve data. In this version, the students subculture from the overnight samples and then assess the banana smell and turbidity (population) of the subcultures every twenty minutes. To increase the number of data points, different classes can measure the same subcultures throughout the day. Alternatively, the subcultures can be refrigerated and warmed back to room temp for 30 minutes prior to the next data point collection. You should expect to collect data over three days to get data points from all phases of the growth curve.

If time allows the students can carry out all parts of the procedure. This adds two class periods in a typical High School AP Biology or Biotechnology class. If instead the teacher prepares in advance the samples as outlined in Part 1 of the procedure, the students can conduct the smell tests and population measurements over two or three days. In a college lab course, most of the bacterial growth curve can be conducted in a typical three to four hour lab period.

This procedure includes instructions for using a spectrophotometer to measure the population growth. If a spectrophotometer is not available, the population can be easily measured using the McFarland Turbidity methodology, as explained below.

Workflow Protocol B

Classroom Content

  • BioBuilder material that sets up this lesson starts here
  • Day 1: streak strains from stabs onto plates
  • Day 2: grow strains from plates as liquid overnights
  • Day 3: subculture bacteria in larger volumes, collect data through lag phase
  • Day 4: subculture continues, collect data through log phase
  • Day 5: finish growth curve, collect data for stationary phase

When you are done with this lab, ask your students to submit their data [here.]

Annotated Procedure

Image:Note mini.png TEACHERS: Clean-up instructions. Provide containers at each work stations for student biological waste such as pipet tips, eppendorf tubes, spreaders, innoculating loops, and plates. Be sure to follow hazardous waste procedures as set forth by your school or municipality. Generally, it is safe to soak the material in each container with a 10% bleach solution for 2 hours. Materials can then be discarded into the regular trash. You can find more information about microbiology lab safety here

Day 1:

TO DO

  • Streak out strains from stabs to plates
  • Prepare banana extract standards
Streak out strains from stabs to plates

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.

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

A video of this procedure is here.
Image:Note mini.png TEACHERS: You may wish to conduct this procedure yourself. However, if time allows, the students will enjoy learning these microbiological techniques. If your class will test the whole set, there will be 4 strains to streak out. Strains can also be streaked out on LB+Amp+Cam if you'd like to verify the indole- strain background.

Prepare banana extract standards

The banana extract is provided in the kit. It will be necessary to make up the standards following the table. Image:Banana_extract_formula.JPG‎
Image:Note mini.png TEACHERS: The banana extract is an oil and will not dissolve in water. However, the concentrations are low and as long as the standard is given a shake before smelling, a suspension is sufficient.

Day 2:

TO DO:

  • Grow liquid overnights of bacterial strains
  • Prepare Turbidity standards (if no spectrophotometer is available)
Grow liquid overnights of bacterial strains
  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 and 3 μl of ampicillin. This volume is more than enough for each strain that each student or team of students must grow.
  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.

A video of this procedure is here.
Image:Note mini.png TEACHERS: If you do not have a roller wheel and an incubator, you can increase the volumes of each culture to 10ml and grow them in small erlenmeyer flasks with stir bars stirring them slowly on a stir plate. Cultures are stable and active for a week at least (stored in the fridge) but will take considerably longer to start growing on the day you subculture (~3 hours rather than 1).

Prepare turbidity standards

As the populations of bacteria increase, the culture media gets increasingly turbid. Using the McFarland Turbidity Scale, it is possible to estimate the changes in turbidity. The results will not be as precise as what you would measure with a spectrophotometer, but the changes over time will be detected and the results can be graphed.


Image:Note mini.png TEACHERS: These standards can be prepared well in advance of lab and are useful if you are running the protocols without access to a spectrophotometer.

Day 3: Measuring bacterial population growth in lag phase

TO DO:

  • Innoculate large volumes and collect data for lag phase

Image:Note mini.pngTEACHERS: The procedure assumes each lab group will measure all 4 cultures. Due to equipment constraints, you may wish to vary this experiment so larger "class size" cultures are grown. In that case, increase the solutions and the amount of bacteria added by a factor equal to the number of lab groups. Students can then remove aliquots from these larger cultures for analysis.

Procedure, if using a spectrophotometer

A video of this procedure is here.
Image:Note mini.png TEACHERS: While isoamyl alcohol is safe enough for lab use, it is best if this is added by the teacher.

  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 spectrophotometer, 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.Image:Note mini.png TEACHERS: If you are using a small plastic cuvette, a 1 ml sample will be sufficient.

  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. Cover the flasks with foil and start them gently stirring on the stir plates.
  7. 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).
  8. Prepare the spectrophotometer by setting it to OD600.
  9. 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. That time will be your T0.Image:Note mini.png TEACHERS: Given time constraints in most high school lab classes, it is OK for the students to prepare these samples and then place them in the refrigerator. The teacher can then place the samples on the room temperature stir plate the next morning and note the time. This may not be necessary during an extended lab period. At room temperature, it will take around three hours for the cultures to enter log phase. Initial readings will have an OD 600 around 0.05.
  10. Add a stir bar to each culture flask and place onto stir plates. Stir slowly. Cover the flasks with foil.
  11. 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. Image:Note mini.png TEACHERS: 1 ml is sufficient for a small plastic cuvette, while 2 mls will be needed for a small test tube that fits into most Spec 20 spectrophotometers
  12. Read the blank and adjust the % Absorbance to zero.
  13. Read the sample tubes and record the % Absorbance.
  14. 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.
  15. At 20 minute intervals repeat steps 11-14.
  16. Between time points, you can calculate the bacterial population: 1 OD600 unit = 1 x 109 bacteria.

Image:Note mini.png TEACHERS: If you are dividing the growth curve into several short lab periods, be sure to store the cells in the fridge (~4°) until the next session.
Image:Note mini.pngTEACHERS: Students will note that the banana smell dissipates a bit while the tube is open. They can close the tube for a minute and then re-shake to bring back the smell.
Image:Note mini.pngTEACHERS: The students will probably notice the subjective nature of smell as they argue over the values. You may want to encourage each group to select a designated "smeller" in order to increase consistency. However, do encourage all the students to take a whiff. The qualitative nature of the banana assay will lead to interesting discussions about the nature of data.

Procedure, if no spectrophotometer is available

Image:Note mini.pngTEACHERS: You may want to have some groups use the spec 20s while others use the McFarland standards. This will lead to interesting comparisons and discussions.
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.

Preparations of McFarland Standards:

Note: Your teacher may instruct you to do this in advance of the labs.

  1. 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.Image:Note mini.png TEACHERS: The size of the tubes and the volume of sample and standard used is flexible. The important things are that the volume can obscure the thick black lines and that the samples and standards are prepared in the same fashion, as shown in the photo.
  2. Place the samples 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.
  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. 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. 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.
  4. Repeat the addition of 2ml of bacteria to 75 ml of broth in the erlenmeyer flasks for each of the overnight cultures.
  5. Add a stir bar to each culture flask and place onto stir plates. Stir slowly. Cover the flasks with foil.
  6. 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.
  7. 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.) That time will be your T0.To take this initial reading, you will compare your cultures to the standards. 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.
  8. 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.
  9. Use the table below to determine the comparable OD 600. 1 OD 600 unit equals approximately 1 x 109 cells.
  10. After 20 minutes repeat steps 6-9, but record the time as T0 + 20.
  11. 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.
  12. At 20 minute intervals repeat steps 6-11. Record time as T0 + the number of minutes since T0.
  13. Your teacher will inform you of the length of this test and may provide you with data from other classes.

Image:Note mini.pngTEACHERS: It is likely that each stationary phase samples will have a turbidity greater than McFarland standard 7. The students can use 7 as the value in that case. You may advise the students that they can use half values (e.g. 1.5). This will also lead to interesting questions about the nature of data.

Day 4: Measuring bacterial population growth in log phase

TO DO:

Restart cultures and collect data for lag phase

Image:Note mini.png TEACHERS: A successful time course for Day 4 could be: Start the samples up in the morning for a class later in the day. These hours will allow the population to reach log phase. At this point, the students can take 2 or 3 more readings, as described for Day 3. The culture can be run overnight at room temperature to reach stationary phase for Day 5

Day 5: Measuring bacterial population growth in stationary phase

TO DO:

Collect data for stationary phase

Image:Note mini.png TEACHERS: Two or three readings from this phase will provide sufficient data points for construction of the population growth curve. Just make sure that the students are tracking the time accurately from the initial reading.

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