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 1031 worldwide, they are
everywhere *from the seas to the soil we walk on*
• One of the most diverse things on the planet
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
4. What are we trying to do?
• We are trying to isolate a new strand of arthobacter
phage by taking samples from the environment
around us.
• 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,
heath, environmental and ecological applications.
5. 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
6. Methods (continued)
• Most of the phage that we got did not yield plaques so we used the Hudson
phage instead (KK 09-17-2013, Enrich 10-3 Hudson – MacKenzie, KK 07-172013, 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
7. 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 with yielded nothing
• We then purified our phage three times using the titration process
•
•
•
•
•
We picked a plaque
Put it into a microcenrifuge tube with PB
(MacKenzie) tittered it out to 10-2 every time , (Sullivan) tittered it out 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
8. 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
9. 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)
10. 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
11. 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
12. 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
13. 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
14. MacKenzie’s Phage
• Coordinates where I got the phage: Lat 44.850554, Long -92.624617
• Location where I got the phage: A few feet from the river at the southern
end of campus
• Time and date collected- 11:30 AM September 9th, 2013
• Soil collection- moist and rich
• Soil depth- 57.2516 mm
• Air temp- 76 degrees F
• Weather conditions- Partly cloudy
• Weight of my soil – 1.47 g
15. First titer/streak plate/spot test
• I also ended up purifying the Hudson phage because my phage did not end
up yielding any plaques 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) – 2.42 x 104
pfu/ml
• The streak plates both had phage (of the same size, so I only continued
with one of them)
16. 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 2.42 x 104 pfu/ml
• 2nd purification (titrations 0 to -2)
• Titer of 4.55 x 104 pfu/ml
• 3rd purification (titrations 0 to -2)
• Titer of 3.6 x 105 pfu/ml
• I got two webbed plates after I picked a plaque from the third purification
17. 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 three 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
4.05 x 107 pfu/ml
18. • This is my first countable plate (-4)
• And my webbed plate (-3)
19. 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 (-2 to -7) and plated it
• The titer that we got from this was 7.92 x 108 pfu/ml
20. 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
.376 and .218, which then calculated out to 0.188 mg/ml)
• On the second test we used the enzymes BamH1, Cla1, EcoR1, Hae111, and
Hind 111 (with 2.7 ml of my DNA)
• On the third test we used the enzymes Pst1, Bcl1, Nco1, and EcoRV (2.16 ml
of my DNA)
24. Sullivan’s Phage
• 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
25. First titer/streak plate/spot test
• I also ended up purifying the Hudson phage because my phage did not end
up yielding any plaques 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 104
pfu/ml
26. 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 104 pfu/ml
• 2nd purification (titrations 0 to -4)
• Titer of 1.728 x 106 pfu/ml
• 3rd purification (titrations 0 to -4)
• Titer of 7.2 x 103 pfu/ml
27. 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 1010 pfu/ml
28. • This is my first countable plate (-5)
• And my webbed plate (between -4 and -5)
• This is my -6 also
29. 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 109 pfu/ml
30. 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)
34. Conclusions
• Mackenzie’s plaques looked like Gordon and Sullivan’s look like Jawnski
• We may have found new phage, but not for sure because our plaques do
look like other known phages plaques
