Musack PhageMusack Phage

By: Ashley JohnsonBy: Ashley Johnson

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o Phage are viruses that infect Phage are viruses that infect bacteria bacteria

o Phage are being looked at with Phage are being looked at with the hope that they can be used the hope that they can be used to treat antibiotic resistant to treat antibiotic resistant bacteria bacteria

o Play important roles in ecologyPlay important roles in ecologyo Affects how organic matter is used Affects how organic matter is used

in an eco systemin an eco system

o Also is a way to change the DNA of Also is a way to change the DNA of bacteriabacteria

o According to the American According to the American Society of Microbiology’s Society of Microbiology’s “Journal of Bacteriology”, “Journal of Bacteriology”, because of the fact phage do because of the fact phage do have such a potential impact have such a potential impact research has shifted so that research has shifted so that scientists are studying a broad scientists are studying a broad range of phage in “real life range of phage in “real life situations” and not just a few situations” and not just a few samples in a labsamples in a lab

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oWas officially Was officially discovered by Felix discovered by Felix d'Herelle in Parisd'Herelle in Paris

oWas used to treat Was used to treat abscesses, acute and abscesses, acute and chronic infections of chronic infections of the upper respiratory the upper respiratory tract, and mastoid tract, and mastoid infection and other infection and other conditionsconditions

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o Objective:Objective: to discover a to discover a unique phage to send in for unique phage to send in for sequencing. The phage they sequencing. The phage they collect with different genomic collect with different genomic sequences are put into an sequences are put into an online database to compare online database to compare to each other. to each other.

o Hypothesis: Hypothesis: If we use If we use standard biological standard biological techniques in the laboratory, techniques in the laboratory, then we will discover a then we will discover a unique phage to send in for unique phage to send in for sequencing. sequencing.

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o Collected Soil SampleCollected Soil Sample

o We collected soil samples with We collected soil samples with the hopes that these soils the hopes that these soils samples would harbor phage.samples would harbor phage.

o Enriched Soil SamplesEnriched Soil Samples

o We enriched the soil samples to We enriched the soil samples to amplify the number of phage amplify the number of phage that we hoped was already in that we hoped was already in the soil samplethe soil sample

o Harvested PlaquesHarvested Plaques

o Once the soil samples were Once the soil samples were enriched we tried to grow enriched we tried to grow plaques to see if we did have plaques to see if we did have phage and to start the purifying phage and to start the purifying process as wellprocess as well

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Con. oPurified PlaquesPurified Plaques

oWe purified plaques so We purified plaques so that we were sure that that we were sure that we continued to work we continued to work with only one phage at with only one phage at a time. a time.

o Purified by streaking for Purified by streaking for isolation and by titrating isolation and by titrating picked plaques outpicked plaques out

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oHarvested Plate LysatesHarvested Plate Lysates

o We did this so that we had We did this so that we had a concentrated sample of a concentrated sample of phage that we could do phage that we could do further tests onfurther tests on

o Isolate and Purify Phage Isolate and Purify Phage DNADNA

o We had to isolate the We had to isolate the phage DNA from the phage DNA from the proteins that made up the proteins that made up the rest of the phage so that rest of the phage so that we could then send it for we could then send it for sequencing and we had to sequencing and we had to purify it so that it was just purify it so that it was just the phage DNA that was the phage DNA that was sentsent

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oQuantify DNAQuantify DNA

oWe had to quantify the We had to quantify the DNA so that we knew DNA so that we knew how much DNA we how much DNA we actually had in the actually had in the sample that we ended sample that we ended up with after isolating up with after isolating and purifying.and purifying.

oWe needed to know how We needed to know how much DNA we had much DNA we had because the digestions because the digestions required a certain required a certain amount of DNA plus amount of DNA plus sequencing did too. sequencing did too.

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o Digest DNADigest DNA

o We digested the DNA so that We digested the DNA so that we could compare it to the we could compare it to the known arthrobactor phage known arthrobactor phage that are in the databasethat are in the database

o We wanted to do this so that We wanted to do this so that we could see if any of our we could see if any of our phage were similar to what phage were similar to what was already known. This was already known. This might suggest that our phage might suggest that our phage was related. was related.

o Analyzing DNA with Electron Analyzing DNA with Electron MicroscopyMicroscopy

o Used an electron Microscope Used an electron Microscope to be able to view our phage to be able to view our phage and to take a picture of it as and to take a picture of it as well. well.

o We were able to tell the size of We were able to tell the size of our phage as wellour phage as well

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o The name of my phage is The name of my phage is Musack. The reason that I Musack. The reason that I picked this name is because I picked this name is because I am engaged and Musack will be am engaged and Musack will be my married last name. Musack my married last name. Musack is not a very common name but is not a very common name but still not too complicated of a still not too complicated of a name either. Plus I thought it name either. Plus I thought it was a cool way to honor my new was a cool way to honor my new life with my soon to be husband life with my soon to be husband and his family. and his family.

o The soil sample that I collected The soil sample that I collected did not produce any plagues, did not produce any plagues, even after re-enrichment. This even after re-enrichment. This phage came from a plaque that I phage came from a plaque that I picked from the “Hudson phage” picked from the “Hudson phage” plate that was labeled 10^-2. plate that was labeled 10^-2. My initials are on the plaque My initials are on the plaque that I picked on that plate.that I picked on that plate.

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ck oPlaque size: Plaque size: Relatively smallRelatively small

oMy HTL titer was My HTL titer was 4.285×10^9 pfu/mL4.285×10^9 pfu/mL

oMy MTL titer was My MTL titer was 3.285×10^9 pfu/mL3.285×10^9 pfu/mL

Spot Test

Webbed plate Countable Plate

My Phage DNA

Gel from first enzyme digestion

Gel determining presence of DNA and quality of DNA

1.Ladder2.Undigested

DNA3.BamHI4.ClaI5.EcoRI6.HaeIII7.HindIII

1 2 3 4 5 6 7

Nco I

Gel from second enzyme digestion

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o Yield of DNA extraction was .1095 Yield of DNA extraction was .1095 µg/µL within a 100 µL sampleµg/µL within a 100 µL sample

o The absorption rating in 260 The absorption rating in 260 light was .219light was .219

o The absorption rating in 280 The absorption rating in 280 it was .136 for a ratio of 1.610 it was .136 for a ratio of 1.610

o My phage, when cut with Nco My phage, when cut with Nco I, looks similar to the phage I, looks similar to the phage Bernie and the phage Bernie and the phage Salgoto. They both have the Salgoto. They both have the double band in about the double band in about the right place and in Salgoto right place and in Salgoto there is a multiple banded there is a multiple banded section towards the end that section towards the end that looks similar to the end of the looks similar to the end of the banded region in my gel.banded region in my gel.

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Phage

Phage oDiameter of head : 40 nmDiameter of head : 40 nm

o Length of tail : 110 nmLength of tail : 110 nm

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o I strongly suggested I strongly suggested for my phage not to for my phage not to be picked for be picked for sequencing.sequencing.

o I enjoyed this class I enjoyed this class because I received because I received hands on experience hands on experience working with a real working with a real life issue while in an life issue while in an environment where I environment where I could learn. could learn.

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Work

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Bruttin, Anne. “Phage-Host Bruttin, Anne. “Phage-Host Interaction: an Ecological Interaction: an Ecological Perspective.” Perspective.” Journal of Journal of Bacteriology Bacteriology 168.12 (2004): n. pag. Web. 10 Dec. 2013.<http://jb.asm.org/conhttp://jb.asm.org/content/186/12/3677.full>tent/186/12/3677.full>

Sulakvelidze, Alexander. Sulakvelidze, Alexander. “Bacteriophage Therapy.” “Bacteriophage Therapy.” n.d. Web. 10 Dec. 2013. n.d. Web. 10 Dec. 2013. <http://www.ncbi.nlm.nih.go<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC90351/>v/pmc/articles/PMC90351/>