PhageBy MacKenzie Miller and Sullivan Mann

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

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

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.

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

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 07-17-2013, 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

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

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

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)

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

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

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

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

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

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)

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

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 myplates that were titratedout

• My plates ended up beingat titer of 4.05 x 107 pfu/ml

• This is my first countable plate (-4)• And my webbed plate (-3)

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

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)

My DNA fragment

DNA Ladder

DNA Ladder

Hind111

Hae111

EcoR1

Cla1

BamH1

My DNA

DNA Ladder

EcoRV

Nco1

Bcl1

Pst1

My DNAMixture

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

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

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

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 myplates that were titratedout

• My plates ended up beingat titer of 1.202 x 1010 pfu/ml

• This is my first countable plate (-5)• And my webbed plate (between -4 and -5)• This is my -6 also

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

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)

My DNA fragment

DNA Ladder

DNA Ladder

Hind111

Hae111

EcoR1

Cla1

BamH1

My DNA

DNA Ladder

EcoRV

Nco1

Bcl1

Pst1

My DNAMixture

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