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  1. 1. Phage Final Report By Sullivan Mann
  2. 2. What is a Bacteriophage? • A virus that parasitizes a bacterium by infecting it and reproducing inside it • Bacteriophage means “eater of bacteria” • There are an estimated 10^31 worldwide, they are everywhere • One of the most diverse things on the planet • Can survive in almost any environment • Very specific, targeting only one or few strands of bacteria
  3. 3. How are they useful? • They destroy up to 40% of the bacteria in Earth’s oceans each day. • The can affect bacteria which can be good and bad • Good – Phage Therapy, the therapeutic use of bacteriophages to treat pathogenic bacterial infections that do not respond to conventional antibiotics (Phage Therapy was mainly used/developed in former Soviet Union circa 1920) • Bad – Phage can shuttle genetic sequences among different bacterial species and strains which can be harmful to people – Can insert their own genes into bacteria and change the genome of the
  4. 4. Methods • Collected our soil samples • Enriched our sample by adding sterile water, sterile 10 x LB medium, and 1 M CaCl solution then we incubated at 37 degrees for 24-48 hours. • We then used this solution and put it in a conical tube, spun it for 2000 rpm for 5 min, then we pour the supernatant into a filtered syringe and pushed what we could through. • We then used 100 ml solution to make a titration out to 10^-4 • Using the titrations we made plates • Incubated at 30 degrees C for 24-48 hours
  5. 5. Methods (continued) • Most of the phage that we got did not yield plaques so we used the Hudson phage instead (KK 07-17-2013, Enrich 10-3 Hudson – Sullivan) • Using an inoculating loop we picked a plaque then put it into a microcentifuge tube with PB and then we preformed the titration process out to -5 • Then using the titrations we plated it out again with the mixture of our phage, host bacteria and TA and incubated at 30 degrees C for 24-48 hours • We also tried to re-enrich our samples and did a spot test to try and get a plaque to see if we had phage, which didn’t work
  6. 6. Methods (continued) • We then made a streak plate with the Hudson phage using an inoculating loop we picked a plaque and streaked it across a plate and poured a TA and arthro mixture onto the least concentrated section first and swirled it carefully from the least concentrated section to the most concentrated section to reduce contamination • We did a spot test of the new enrichment which yielded nothing • We then purified our phage three times using the titration process • We picked a plaque • Put it into a microcentrifuge tube with PB • Titered it out (Sullivan to -4, -3, then -2) • Placed it in a corresponding labeled host bacteria and let sit for 15 min • Added TA and poured them onto plates
  7. 7. Methods (continued) • Using a webbed plate we added 8ml of PB onto it and let sit for an hour • We then filtered the PB/phage solution using a filter syringe • Then using that we titrated out to 10^-10 • And using the titrations we plated all of them out with the phage/host bacteria/TA mixture • Also using the titration we did a spot test where we put 5 ml of each titration onto a different square • Then we incubated them at 30 degrees C
  8. 8. Methods (continued) • Using the titration number that was best webbed plate we took our MTL and titrated it out to that number again. • Then using that number we took that titration and plated out 10 plates (using the phage/bacteria/TA mixture) and incubated at 30 degrees C • This then yielded 10 webbed plates which we flooded with PB and let sit for an hour • Then we pipetted the PB/phage mixture into a conical tube and spun for min @ 2200 rpm • Then using a vacuum filter we filtered the supernatant which was then the 100 (HTL)
  9. 9. Methods (continued) • We then did a titration of our HTL to find the titer of the HTL • We also isolated the DNA by putting some of our phage into an oak ridge tube, adding nuclease mix and mixing it by inversion, then we incubated at 37 degrees C for 30 min. • Then we let it sit for an hour at room temp. • Then we added phage precipitant to the nuclease treated lysate and mixed by inversion and incubated at 4 degrees C
  10. 10. Methods (continued) • Using our phage in the oak ridge tubes we spun them in a centrifuge for 20 min at 10,000xg • Then we poured out the supernatant (not disturbing the pellet at the bottom) and drained the excess liquid by inverting the tube and letting it sit for 2-3 min • Then we added sterile water and gently re-suspended the pellet and let sit for 5-10 min • Then we added pre-warmed DNA clean up resin and then uncoated the phage by pipetting the mixture up and down and swirling the tube
  11. 11. Methods (continued) • Then using 2 columns we added some of our solution to each using a pipette and then use a plunger to push the solution the solution through • For each column we then pushed isopropanol through to wash the salts and proteins off the DNA • Then we dried the columns by centrifuging them at max speed for 5 min, then we transferred the columns to a new tube without lid and centrifuged if for a min at max speed • Then we transferred it to a new tube, added pre-warmed (80 degrees C) TE to the resin in the column and let sit for a min and then centrifuged at max speed for a min • We combined the DNA into a single tube and stored at 4 degrees C
  12. 12. Methods (continued) • We ran some of our DNA through a spectrometer to calculate our micrograms per microliter • We made gels using agarose, 1 x TAE buffer, and gel red • We then electrophoresed 3 different gels • The first gel was just our DNA vs. a DNA ladder • The second gel was mixing our DNA with the enzymes BamH1, Cla1, EcoR1, Hae111, Hind111, our DNA by itself and a DNA ladder (to see which enzyme would cut our DNA) • The third gel was mixing our DNA with the enzymes Pst1, Bcl1, Nco1, EcoRV with a DNA ladder • For the enzyme mixtures we used a mixture of 10x reaction buffer, our DNA, 10x BSA, the enzyme and sterile water
  13. 13. Sullivan’s Phage: JJFlash13 • Coordinates where I got the phage: Lat 45.277801, Long -92.015927 • Location where I got the phage: My house from under a board • Time and date collected- 1:30 PM September 8th, 2013 • Soil collection- dry and loose • Soil depth- 55 mm • Air temp- 74 degrees F • Weather conditions- Cloudy, humid, occasionally misting • Weight of my soil – 2.49 g
  14. 14. First titer/streak plate/spot test • I also ended up purifying the Hudson phage because my soil did not end up yielding any phage the first try (or when we tried to re-enrich it) • Titer of the first set of plates (which went out from 0 to -5) – 2.48 x 10^4 pfu/ml
  15. 15. Purification (3 times) • After picking the streak plate and plating it from 0 to -2, I got phage on all of my plates (with numbers that consecutively went down as the dilution went up), with two different sized plaques • 1st purification (titrations 0 to -5) • Titer of 2.48 x 10^4 pfu/ml • 2nd purification (titrations 0 to -4) • Titer of 1.728 x 10^6 pfu/ml • 3rd purification (titrations 0 to -4) • Titer of 7.2 x 10^3 pfu/ml
  16. 16. Phage Lysate • We made a spot plate, and we also picked a plaque from the last purification and titrated out from 0 to -10 and I got four webbed plates and three countable plates • This was the spot plate • It corresponded to my plates that were titrated out • My plates ended up being at titer of 1.202 x 10^10 pfu/ml
  17. 17. • This is my first countable plate (-5) • My webbed plate (between -4 and -5, not shown) • This is my -6 also
  18. 18. HTL • Using the webbed plates we flooded them and plated them on ten new plates, which then yielded 10 webbed plates. We then collected a mixture of phage and PB and filtered it (our HTL and the new 100 ) • We also did a titration of this HTL (-3 to -8) and plated it • The titer that we got from this was 3.2 x 10^9 pfu/ml
  19. 19. Electrophoresis • Using the HTL we isolated the DNA from anything else that had been in our mixtures. • We then mixed this isolated DNA with different enzymes so we could run electrophoresis • On the first test we had a group of 4 using one gel just using the DNA (2 ml of it) • We also ran the DNA through a spectrophotometer and got the values . 427 and .240, which then calculated out to 0.2135 mg/ml) • On the second test we used the enzymes BamH1, Cla1, EcoR1, Hae111, and Hind 111 (with 2.3 ml of my DNA) • On the third test we used the enzymes Pst1, Bcl1, Nco1, and EcoRV (1.8 ml of my DNA)
  20. 20. A A= DNA Ladder B= My DNA fragment B
  21. 21. A= DNA Ladder DNA B= My C= BamH1 D= Cla1 E= EcoR1 F= Hae111 G= Hind111 A B C D E F G
  22. 22. A MacKenzie's DNA Mixture A-E My DNA Mixture F= DNA Ladder G= Pst1 H= Bcl1 I= Nco1 J= EcoRV B C D E F G H I J
  23. 23. Conclusion: What we are doing • The class was trying to isolate a new strand of arthrobacter phage. We were able to do that by taking samples from the environment around us and using procedures, techniques and tools provided to us. • The goal of this experiment is to learn to think like a scientist and to discover new strands of phage. • This could provide a framework for SEA scientists and other researchers to delve into the possible utility of these organisms in a variety of biomedical, health, environmental and ecological applications.