Musack Phage By: Ashley Johnson

Intro o Phage are viruses that infect bacteria o Phage are being looked at with the hope that they can be used to treat antibiotic resistant bacteria o Play important roles in ecology o Affects how organic matter is used in an eco system o Also is a way to change the DNA of bacteria o According to the American Society of Microbiology’s “Journal of Bacteriology”, because of the fact phage do have such a potential impact research has shifted so that scientists are studying a broad range of phage in “real life situations” and not just a few samples in a lab

Intro Con. o Was officially discovered by Felix d'Herelle in Paris o Was used to treat abscesses, acute and chronic infections of the upper respiratory tract, and mastoid infection and other conditions

Intro Con. o Objective: to discover a unique phage to send in for sequencing. The phage they collect with different genomic sequences are put into an online database to compare to each other. o Hypothesis: If we use standard biological techniques in the laboratory, then we will discover a unique phage to send in for sequencing.

Methods o Collected Soil Sample o We collected soil samples with the hopes that these soils samples would harbor phage. o Enriched Soil Samples o We enriched the soil samples to amplify the number of phage that we hoped was already in the soil sample o Harvested Plaques o Once the soil samples were enriched we tried to grow plaques to see if we did have phage and to start the purifying process as well

Methods Con. o Purified Plaques o We purified plaques so that we were sure that we continued to work with only one phage at a time. o Purified by streaking for isolation and by titrating picked plaques out

Methods Con. o Harvested Plate Lysates o We did this so that we had a concentrated sample of phage that we could do further tests on o Isolate and Purify Phage DNA o We had to isolate the phage DNA from the proteins that made up the rest of the phage so that we could then send it for sequencing and we had to purify it so that it was just the phage DNA that was sent

Methods Con. o Quantify DNA o We had to quantify the DNA so that we knew how much DNA we actually had in the sample that we ended up with after isolating and purifying. o We needed to know how much DNA we had because the digestions required a certain amount of DNA plus sequencing did too.

Methods Con. o Digest DNA o We digested the DNA so that we could compare it to the known arthrobactor phage that are in the database o We wanted to do this so that we could see if any of our phage were similar to what was already known. This might suggest that our phage was related. o Analyzing DNA with Electron Microscopy o Used an electron Microscope to be able to view our phage and to take a picture of it as well. o We were able to tell the size of our phage as well

Musack o The name of my phage is Musack. The reason that I picked this name is because I am engaged and Musack will be my married last name. Musack is not a very common name but still not too complicated of a name either. Plus I thought it was a cool way to honor my new life with my soon to be husband and his family. o The soil sample that I collected did not produce any plagues, even after re-enrichment. This phage came from a plaque that I picked from the “Hudson phage” plate that was labeled 10^-2. My initials are on the plaque that I picked on that plate.

Musack o Plaque size: Relatively small o My HTL titer was 4.285×10^9 pfu/mL o My MTL titer was 3.285×10^9 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.Ladder 2.Undigested DNA 3.BamHI 4.ClaI 5.EcoRI 6.HaeIII 7.HindIII

Nco I Gel from second enzyme digestion

Musack o Yield of DNA extraction was.1095 µg/µL within a 100 µL sample o The absorption rating in 260 light was.219 o The absorption rating in 280 it was.136 for a ratio of o My phage, when cut with Nco I, looks similar to the phage Bernie and the phage Salgoto. They both have the double band in about the right place and in Salgoto there is a multiple banded section towards the end that looks similar to the end of the banded region in my gel.

Measurements of Phage o Diameter of head : 40 nm o Length of tail : 110 nm

Conclusion o I strongly suggested for my phage not to be picked for sequencing. o I enjoyed this class because I received hands on experience working with a real life issue while in an environment where I could learn.

Work Cited Bruttin, Anne. “Phage-Host Interaction: an Ecological Perspective.” Journal of Bacteriology ent/186/12/3677.full> Bruttin, Anne. “Phage-Host Interaction: an Ecological Perspective.” Journal of Bacteriology (2004): n. pag. Web. 10 Dec Sulakvelidze, Alexander. “Bacteriophage Therapy.” n.d. Web. 10 Dec Sulakvelidze, Alexander. “Bacteriophage Therapy.” n.d. Web. 10 Dec