Test phages for broad host range

Objective

To determine if any of the phages we isolated can infect environmental strains of C. glu

Rationale

So far, we have been using a “lab strain” of C. glu called MB001. This strain has been somewhat domesticated to be a good subject for lab study. However, we have discussed that bacteria are all throughout our environment, and C. glu in particular likes to grow in the soil. In these experiments, we are going to test if the phages that we isolated on our lab C. glu are capable of infecting a broad range of C. glu strains including the original ancestor to our lab strain and two different environmental strains.

Materials:

  • High titer lysates of each of your two phages
  • Pre-melted top agar supplemented with 10mM CaCl2 and 0.4% glucose
  • LB agar plates
  • Serological pipettes
  • Microcentrifuge tubes
  • SM buffer
  • Four strains of C. glu

Procedure for making host range plates

Day 1

  1. Get five LB agar plates and label each one with your name, date, and one host strain of C. glu, and draw a grid on it as we did for the spot assay plates before. You should have two MB001 plates, and one of each of ATCC, Env1, and Env2.

    The four host strains of C. glu we are using are called MB001, ATCC, Env1, and Env2. MB001 is the strain we used for the initial screens, ATCC is the ancestor strain of MB001, and Env1 and 2 are the environmental strains.

  2. Label the top of each MB001 plate with one of your phage IDs and the grid for dilutions 10-2 through 10-9. Keep one section for no phage control.
  3. Label each of section on the new host plates with phage ID and dilution. You should have one section with no phage, and four for each of your phages. Follow the picture above for reference.

    We are plating less dilutions on the new host because we expect the phage to have a harder time infecting the new hosts. We only expect to see plaques in the very concentrated spots on the new hosts.

  4. Pour a lawn with the correct strain of C. glu onto each plate. Remember to add 250uL of the correct bacteria and 4mL of top agar. Allow the lawns to set while you complete the next few steps.
  5. Label 9 tubes and make 10-fold serial dilutions of your high titer stocks from undiluted through 10-9. Each tube should have 90uL of SM buffer to start. Transfer 10uL from your undiluted stock through each tube until the end of your dilution series.
  6. Use 5uL drops to spot your dilutions onto each of your plates in the correct sections.
  7. Allow the drops to dry on the bench for at least 10 minutes, and then move to the 30C incubator until tomorrow.

Day 2

  1. Observe if you got plaques on the lab strain MB001 as expected and if you got any plaques on the other three strains. Record your observations in your lab notebook.
  2. Choose a plaque from one of the new strains to pick and study further. Circle it on your plate and then scan your plates for future reference.
  3. Give this phage a new phage ID. This phage ID should be your previous phage ID + mut. For example, a plaque picked from Ellie’s phage EAR P2, will be called EAR P2mut to tell us it is a mutated version of EAR P2.
  • Titer your plaque on all strains

Here we will see how well your new, mutated phages can infect the range of C. glu host strains.

  1. Get four new LB agar plates and label with your name, date, and one host strain of C. glu. Draw a grids on each plate and label the sections for a titer plate. Leave one section for no phage control, one for undiluted plaque, and the rest for serial dilutions through 10-7.
  2. Pour a lawn with the correct strain of C. Glu onto each plate. Remember to add 250uL of the correct bacteria and 4mL of top agar. Allow the lawns to set while you complete the next few steps.
  3. Fill a microcentrifuge tube with 500uL of SM buffer.
  4. Pick your plaque of interest and add it to the SM buffer.
  5. Label 7 more tubes and make 10-fold serial dilutions of your plaque through 10-7. Each tube should have 90uL of SM buffer to start. Transfer 10uL from your undiluted stock through each tube until the end of your dilution series.
  6. Use 5uL drops to spot your dilutions onto each of your plates in the correct sections.
  7. Allow the drops to dry on the bench for at least 10 minutes, and then move to the 30C incubator until tomorrow.
  • Plate your plaque for webbed plate

Here we will make webbed plates for high titer lysates like we did for your original phages.

  1. Get three new LB agar plates and label them with your name, date, the host you picked your plaque from, new phage ID, and a dilution factor. The dilution factors for this experiment are undiluted, 10-1, and 10-2.
  2. Get three culture tubes, and label them with host and dilution factor corresponding to your plates.
  3. Add 250uL of your host into each tube.
  4. Using the plaque solution and dilutions you made above, add 100uL of the correct concentration into each tube.
  5. Let the cells and phage incubate together on the bench for 10 minutes.
  6. Use 4mL of top agar to pour your lawns.
  7. Allow the plates to solidify for at least 10 minutes, and then move to the 30C incubator until tomorrow.

Day 3

  • Titer plates
  1. Observe if you got plaques on the same strain as yesterday and if you got plaques on any new strains.
  2. Calculate the titer on all four strains.
  3. Record your observations and titers in your lab notebook.
  4. Scan your plates to keep for your reference.
  • Webbed plates
  1. Scan your plates to keep for your reference.
  2. Pick which plate has the best webbed plaques and add 4mL of SM buffer to the top.
  3. Allow to incubate over the weekend.

Day 4

  1. Label two 15mL tubes with your new phage ID, name, and date.
  2. Carefully pipette the SM buffer off the plate into one of 15mL tubes.
  3. Use a syringe and corresponding filter to filter the SM buffer into the second 15mL tube. This will be your high titer lysate for your mutated phage.
  4. Get a new LB agar plate. Label it with your name, date, new phage ID, and the host you used for your webbed plates. Make a grid on it to plate serial dilutions including no phage control, undiluted, and 10-1 through 10-7.
  5. Pour a lawn using your host strain of C. glu. Allow it to set while you make your serial dilutions.
  6. Get 7 microcentrifuge tubes and label them 10-1 through 10-7.
  7. Add 90uL of SM buffer into each tube, then transfer 10uL of your high titer lysate into the first tube.
  8. Continue making serial dilutions through 10-7.
  9. Use 5uL drops to spot your dilutions onto each of your plates in the correct sections.
  10. Allow the drops to dry on the bench for at least 10 minutes, and then move to the 30C incubator until tomorrow.

Day 5

  1. Observe your plate and calculate the titer of your new high titer stock.
  2. Record your observations and titer in your lab notebook.
  3. Scan your plates to keep for your reference.
  4. Use your new high titer stock for down stream experiments such as extracting the phage genomic DNA.