Phage

1. Phage By: Ethan and Noah

2. Purpose • To collect, recreate and analyze phage from the environment • To sequence DNA from phage

3. What is a phage? • Virus • Smaller than bacteria • Found almost anywhere

4. Phage Therapy • Using known phage to cure bacterial infections • Sometimes involves “reprogramming”

5. My Phage • Collected from base of tree in River Falls, WI • Taken from moist soil conditions • About 44mm below surface • Named Lola13

6. Enrichment of Environmental Samples • Materials: Soil, Sterile water, Sterile 10 x LB medium, calcium chloride, and bacteria • Procedure: • Soil was collected around Hudson • The soil was weighed out and added to a flask • Aseptically, water, LB medium, CaCl2 and bacteria was added, and then incubated at 37°C for approximately 48 hours

7. Harvesting and Preparing the Enriched Sample • Materials: Soil sample prepared, phage buffer, microcentrifuge tubes, conical tubes, top agar, and agar plates\ • Procedure: • Pour part of the prepared soil sample into a conical tube and centrifuge • Pour the supernatant into a filter unit, making it the 100 sample • Then take four microcentrifuge tubes and add phage buffer to each • Transfer part of your sample into the first tube, and vortex it • Continue this for the rest of the tubes and get tubes of the host bacteria • Transfer your sample into a culture tube and allow to sit in room temperature • Then pour top agar into each culture tube and then transfer the solution onto agar plates and allow them to solidify and then incubate

8. Phage Titer, and Streak Plate • Materials: Sterile inoculating loops, agar plates, and top agar • Phage Titer: • Count the number or plaques on a countable plate • Use the following equation to determine phage titer: • # pfu X 1000 ul x 1 = pfu/ml 50 ul 1ml df • Streak Plate: • Grab an agar plate and divide into a T and number 1-3 • Using an inoculating loop, pick one plaque and spread it into one area • Using a clean inoculating loop, spread a little in area #1 and go into area #2 • Using a clean inoculating loop, spread into area #2 and go into area #3 • Take TA and spread onto the least concentrated area first and then incubate

9. Phage Purification • Materials: Inoculating loop, phage buffer, host bacteria, and top agar • Procedure: • Get microcentrifuge tubes and fill with phage buffer • Using an inoculating loop, pick one plaque and place into a microcentrifuge tube and vortex it • Then transfer part of your sample into the next microcentrifuge tube and vortex it • Continue this for the rest of your tubes • Transfer part into culture tubes and allow to sit • Add top agar to each culture tube and spread out evenly over the agar plates • Repeat this procedure two more times to get a good, purified phage

10. Harvesting a Phage Lysate • Materials: Agar plates, phage buffer, conical tubes, filter system, microcentrifuge tubes, top agar, host bacteria, and inoculating loops • Procedure: • Figure out which plate represents a web plate and add phage buffer to it and swirl it gently and wait for an hour • Collect the phage buffer into a conical tube and label as unfiltered • Filter the sample into a microcentrifuge tube and label as 100 • Grab more tubes and put phage buffer into them and perform dilutions through 1010 • Perform a spot test by dividing up an agar plate, and taking part of each sample in each microcentrifuge tube and putting it on the plate and spreading top agar over it • Then transfer some of each sample into culture tubes and wait. • Add top agar to each tube and spread out onto agar plates evenly

11. Phage Titer and Web Plates • Materials: Top agar, phage buffer, agar plates, microcentrifuge tubes, MTL • Phage Titer: • Count the number of plaques on a countable plate • Use the following equation to determine phage titer: • # pfu X 1000 ul x 1 = pfu/ml 10 ul 1ml df • Web Plates: • Pick which plate looked the most like a web plate • Set up enough dilutions to get to that plate • Then take enough of your sample into ten host bacteria tubes and wait • Add top agar to each tube and then spread out evenly over the ten agar plates

12. Harvest Web Plates • Materials: Phage buffer and vacuum filter • Procedure: • Add phage buffer to each web plate and allow to sit for an hour • Pipette the mixture into a conical tube and centrifuge • Then using a vacuum filter, filter the sample into another tube and properly label both tubes

13. Isolate and Purify Genomic DNA • Materials: Phage buffer, Nuclease Mix, HTL, phage precipitant, top agar, and host bacteria • Procedure: • Transfer some of the filter-sterilized phage lysate into an Oak Ridge Tube and store the remaining lysate at 4°C • Add Nuclease Mix and mix • Incubate for 30 minutes and then let sit at room temperature for an hour • Pipette phage precipitant solution to the nuclease – treated lysate and cap • Mix through inversion and let sit overnight • Phage Titer Procedure • Add phage buffer to enough microcentrifuge tubes and do enough dilutions • Add to culture tubes and let sit • Add top agar and spread out over the agar plates and incubate

14. Isolate and Purify Genomic DNA • Materials: Microcentrifuge tubes, Clean Up Rosin, distilled water, isopropanol and TE • Procedure: • Centrifuge the Oakridge tube and decant the supernatant • Add distilled water to the pellet and resuspend it • Add Clean Up Resin and swirl it • Attach 1 column to a syringe and apply the mixture through the column • Add isopropanol to the column and push through it • Centrifuge the column and apply pre-warmed TE to the resin and let it sit and then centrifuge • Repeat for a second column and then combine the DNA samples into a single tube and store at 4°C • Phage Titer: • Count the number or plaques on a countable plate • Use the following equation to determine phage titer: • # pfu X 1000 ul x 1 = pfu/ml 10 ul 1ml df

15. Gel Electrophoresis and DNA Quantification • Materials: Agarose powder, TAE buffer, gel red, DNA ladder, Loading dye, TE • Gel Electrophoresis: • Add agarose powder to an Erlenmeyer flask and add TAE buffer • Microwave and allow the solution to boil over • Add gel red and pour into the gel electrophoresis plate and allow to harden • Pull out the comb and flood with TAE buffer • Inject a DNA ladder in the first well • In a microcentrifuge tube add TE, DNA, and loading dye • Vortex the solution and inject it into a well • DNA Quantification: • Put a sample of your DNA into a spectrophotometer and record the numbers and calculate out the amount of micrograms per microliter

16. Digest Phage Genomic DNA, Set Up Gel • Materials: Reaction buffer, DNA, enzymes, water, agarose powder, and TAE buffer • Procedure: • First mix your DNA sample by flicking the closed tube and incubate • Prepare each tube according to the table and store until ready • Gel Electrophoresis: • Add agarose powder to an Erlenmeyer flask and add TAE buffer • Microwave and allow the solution to boil over • Add gel red and pour into the gel electrophoresis plate and allow to harden

17. Electrophoresis • Materials: Gel, cut up DNA, loading dye • Procedure: • Add TAE buffer to the gel so that it is over flooded • Add dye to the cut up DNA • Load the DNA ladder and samples 1-6 into the wells • Turn on to 100 volts

18. Digest Phage Genomic DNA • Materials: Reaction buffer, DNA, enzymes, water, agarose powder, and TAE buffer • Procedure: • First mix your DNA sample by flicking the closed tube and incubate • Prepare each tube according to the table and store until ready • Gel Electrophoresis: • Add agarose powder to an Erlenmeyer flask and add TAE buffer • Microwave and allow the solution to boil over • Add gel red and pour into the gel electrophoresis plate and allow to harden

19. Gel Electrophoresis • Materials: Gel, cut up DNA, loading dye • Procedure: • Add TAE buffer to the gel so that it is over flooded • Add dye to the cut up DNA • Load the DNA ladder and samples 1-6 into the wells • Turn on to 100 volts

20. Setting up Electron Microscopy • Materials: Bacteriophage, phage buffer, parafilm, grid, distilled water, uranyl acetate • Procedure: • Transfer some HTL into a sterile microcentrifuge tube and centrifuge for an hour • Remove most of the supernatant and add phage buffer • Remove the cover paper from a piece of parafilm and place into a petri dish • Place a PELCO tab onto the parafilm and remove the liner • Remove 2 grids and place shiny side up so the edge is touching the adhesive • Place part of the phage preparation onto each grid • Wait and then wick off the excessive fluid and then place water onto each grid • Wick off and then place more water onto each grid • Add uranyl acetate to each grid and then wick off and allow to air dry • Transport the grids to an EM facility

21. Results (After 3 Enrichments) • 3 different morphologies developed • Created a streak plate for each • Smaller and bigger morphologies purified very well • Titrated each out separately with 4 dilutions

22. Results (After discovering morphologies) • Smaller morphology had plaques to 2nd dilution • Bigger had plaques to 4th dilution Small titer: 4.6x10^-4 pfu/mL Bigger titer: 5.4x10^-6 pfu/mL • Repeat purification

23. Results (after repeating purification) • Smaller morphology disappeared. • Bigger morphology purified to all plates (8 in 10^-2, 2 in 10^3 and 1 in 10^-4)

24. Spot test to determine web plate Countable plate to determine titer Titer: 2.8x10^-4 pfu/mL

25. Web Plate • Plate webbed very well • Small morphology appeared again • Going to set up 10 web plates

26. Harvest HTL and Titer • All 10 plates completely plaqued out • Titer came out to be 2.75x10^-9 pfu/mL

27. DNA Quantification • Spectrophotometer gave me .119 mg/mL

28. Restrict and analyze phage DNA

29. Restrict and analyze phage DNA

30. Restrict and analyze phage DNA (again)

32. EM Picture • The tails are approximately 70 nm • The heads are approximately 30 nm long

33. Why choose my phage? • I don’t think we should use my phage. My phage was my own sample, but has a surprising resemblance to a Hudson phage.

34. Results - Noah • Names: Pyero because it is a pretty cool name • Phage Titer: The first time I did phage titer I got 2.72 x 104pfu/ml, the second time I got 1.06 x 105pfu/ml, the third time was 5.00 x 103pfu/ml, the fourth time was 1.91 x 108 pfu/ml, and the final time was 6.52 x 1010pfu/ml • I got 1.41 x 10-1ug/ul for my DNA quantification

35. Photos of this Process • This picture is a spot test to help determine which plates would be webbed plates • This picture is my countable plate, which can be used for determining titer

36. Photos of this Process • This is my closest example of a web plate. For creating the web plates, I used 30 ul of the 10-3 • My DNA sample is the sixth well from the bottom of the gel, and is being compared to a DNA ladder

37. Photos of this Process • This is a picture of the gel, being cut with various enzymes. From top to bottom, it goes Hind III, Hae III, Eco R I, Cla I, Bam H I, undigested DNA, and the DNA ladder • This is a picture of the gel, being cut with various enzymes. From top to bottom, it goes Eco R V, Neo I, Bcl I, Pst I, and the DNA ladder

38. EM Pictures • The tails are approximately 60 nm long • The heads are approximately 30 nm long

39. Phage Most Similar To I believe that my phage is most similar to Jawanski – however, there aren’t any real distinctive cuts in mine that I can compare using the program because of the lack of enzymes

40. Why Should We Choose my Phage? • I don’t believe that we should choose my phage • It is one of the Hudson phage, which is common in this classroom • It seems remarkably similar in the gel electrophoresis results

41. Conclusion • Neither of us think our phage should be selected. There is a large possibility we somehow ended up with the same phage. Tail: 60 nm Head: 30 nm Tail: 70nm Head: 30 nm