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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. • This is my first countable plate (-5)
• My webbed plate (between -4 and -5, not shown)
• This is my -6 also
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. 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)
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.
