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Christopher Harness

Dr. S. Conant and J. Thompson

ReBUILD SEA Phages

1 December 2015

Discovering and Identifying Bacteriophages

Medical practices over the past century have led to very beneficial discoveries on curing

diseases that run rampant throughout mankind and other living organisms. One of these modern

practices involves combating bacterial infections through the use of bacteriophage, or viruses

aimed to infect and replicate towards specific bacteria via their receptive genes. An example in

which phage-based therapy is effective against other treatments such as antibiotics is when the

infection is inaccessible through the use of these treatments. According to research performed by

Dr. Stephen T. Abedon and his team, situations where antibiotics perform poorly “include

osteomyelitis, diabetic infections of the feet, burns and infections of the central nervous system,

which can be protected from antibiotics due to the presence of the blood-brain barrier (but which

does not necessarily prevent adequate phage penetration to combat infections)”. In a research lab

performed by student of the University of Detroit Mercy, they were tasked in collecting a soil

sample of any location, in which they would purify, isolate, and identify the sample for possible

bacteriophage held from within. The purpose of this was to learn of research lab etiquette and

how to analyze unknown bacteriophage.

In the SEA Phages lab, the research team of students Christopher Harness and Victoria

Torres collected their soil sample on September 6th, 2015 at 10:36 AM in Christopher’s local

park, Moravian Park of Sterling Heights, Michigan. The approximate location, according to the

GPS reading, was 42.545778 degrees North and 82.975505 degrees West. The sample was

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located in a shaded, forest area when the air temperature was 25℃, while the sample itself was

dark, damp, and dug up from a 13.1763 cm depth below ground level. The sample was then

taken back and underwent an enrichment process in order for it to properly adsorb with the

bacteria, Mycobacterium smegmatis (M. smeg for short). This process had the soil sample mix

with materials such as H2O, 10X 7H9/glycerol broth, and even a small sample of M. smeg in

order to become a proper enrichment sample. From there, this sample underwent serial dilutions

via phage buffer (down to the 10-6 dilution factor) and put on a petri dish plate once it had

properly adsorbed with 0.5 mL of M. smeg and has been solidified by top agar. The results were

successful, each plate having plaque morphologies.

The next process was to create a spot plate choosing plaque morphologies on any

enrichment plate. Four plaques (labeled A – D) where chosen on the 10-1 Enrichment Plate,

having spots B and C contain the most plaques to sample off of for further tests. The chosen spot,

C, underwent a purification process known as “streak plating,” where the chosen sample would

be streaked across a petri dish in order to further isolate given plaques. This process was repeated

up to three times, each new streak was taken from an isolated plaque from the previous plate, but

an isolated plaque was chosen from the second streak plate for further testing due to the third

streak plate having its top agar not solidifying properly.

Having the plaque undergo titer assays furthered the process of isolating and purifying

the sample phage, but was proven to be the most difficult due to reoccurring issues. Although the

first titer assay resulted in consistent plaque morphologies, the second assay (and multiple

disregarded ones) all contained two types of plaques: lysogenic (A) and lytic (B). The

researchers decided to create proceeding titer assays on both morphologies which resulted in the

lysogenic “Plaque A” containing a hybrid lytic-lysogenic plaque population in its next titer

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(whereas B had the same two populations). Another titer assay was performed on this population,

having the 10-3 result in a “webplate” (a plate where it almost fully consists of plaques, but has

enough bacteria present) and become flooded by phage buffer to help create a Medium Titer

Lysate.

Medium Titer Lysate (MTL) contains a purified phage sample, but its concertation (in

plaque count over milliliteror pfu/mL) is determined via spot testing, this time recording the

actual plaque count contained within the range of 5 – 50 plaques within any dilution factor

placement (up to 10-10). The concentration was calculated by the equation:

¿ pfu5 μL

× 1000 μL1mL

× D .Factor ; in this case, the plaque count was 6 located in the 10-5 region,

resulting in the calculation 1.2 ×108 pfu /mL. To create identical and factored webplates, the

amount of MTL needed (in mL) is calculated by the area of the plate over the area of the plaque,

over the MTL concentration. This calculation resulted in 7.0 ×10−3 μL of MTL needed to create a

1X Factor webplate. Out of the plates made, a 5X plate became the only successful web out of

the group. From there, four 5X plates were created for flooding purposes to create a High Titer

Lysate (HTL).

The process to determine the HTL concentration is similar to the MTL’s; however, 10-6 to

10-12 dilution plates are created instead of a single spot plate. Calculations for this concentration

are similar, but instead of ¿ pfu5 μL being in the equation, it is:

¿ pfu50 μL . For this calculation, 17 spots

were found on the 10-6 plate, resulting in 3.4× 108 pfumL being the concentration for this HTL.

From there, the HTL sample undergoes both DNA isolation and purification, and Transmission

Electron Microscopy (TEM Imaging).

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Under the DNA purification process, the DNA concentration totaled out to be 51.5 ngμL .

From there, 9.7 μL of this DNA concertation was exposed to the restriction enzymes: BamHI,

ClaI, EcoRI, and HaeIII (HindIII being left out), to help identify a possible DNA sequence based

on how many fragments were “cut” by the enzymes. These measurements were determined by

the placements of the enzyme cuts under the process of gel electrophoresis. The gels would

display the enzymes distance from the starting “wells” after a period of time has elapsed. This

resulted in the DNA exposed to the HaeIII restriction enzyme having the highest distance from

the well; although, the DNA in this experiment may have been denatured in the purification

process.

As the HTL sample underwent TEM Imaging, curtesy of Wayne State University,

clusters of bacteriophage were identified, all of which shared similar characteristics to the

Siphoviridae type of bacteriophage (only consisting of a present “tail” and head). Upon

completion of this experiment, the researchers nicknamed their phage “Phernando” for the sake

of alliteration (and pun) to the word “phage” and to give it a human-like characteristic after

months of hard work to identify it. This work experience in the lab has allowed them to become

more familiar with the rigorous tasks present in any scientific research lab, and how to

collaborate as a team to properly complete the task at hand. The knowledge obtained could one

day be used in their careers in the medical field whenever they are tasked to cure a possible

illness or to even diagnose/treat a patient who is in great illness from a disease.

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Works Cited:

Abedon, Stephen T et al. “Phage Treatment of Human Infections.” Bacteriophage 1.2 (2011):

66–85. PMC. Web. 30 Nov. 2015.

<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278644/>.

Conant, Stephanie, and Jack Thompson. “SEA Phages.” University of Detroit Mercy. 2015.

PowerPoint. 29 November 2015.