Ms. Frizzle Goes Viral: Discovery and Classification of Bacteriophage "Friz":

Carson McCaskell, Marin EhrmantrautBiochemistry and Biotechnology, Minnesota State University Moorhead, 1104 7th Avenue South, Moorhead, MN 56563

Introduction:Bacteriophages, otherwise known as phages, are the most common

biological entities in nature, and are completely unique to each organism.Phages are viruses that inject their DNA into very specific bacteria toreproduce. These viruses are picky about what bacteria they infect, andthe bacteria they can infect range from a single strain of bacteria, to multiplespecies. The phage that is discussed in this experiment, Friz, was onlydiscovered after the third attempt to find a phage that infects Microbacteriumfoliorum. The virus' DNA "hijacks" the bacteria's natural cellular processes toreplicate and reproduce the virus instead. This experiment took advantage ofthe phage's reproduction to cultivate and study the phages that infect thebacteria Microbacterium foliorum. The reason for the study of phages is tounderstand how they work and how they can be used to fight off commonbacterial infections that affect humans. Although they are very particular inthe bacteria they inhabit, there are billions upon billions of phages in theworld that can be studied, cultivated, and used to save lives.

AcknowledgmentsWe would like to thank the Howard Hughes Medical Institute and the Hatfull Lab at theUniversity of Pittsburgh for providing bacteria, support, and training materials, our professors Dr.Jeffery Bodwin and Dr. Michelle Tigges for their knowledge and support for our phages, and toNDSU for allowing our team to use their electron microscope to see and classify our phage.

Objectives:Our goal for this project is to isolate and cultivate a new phage in the

hopes of furthering research and understanding of bacteriophages through isolation, purification, amplification, and continued discovery and research of our individual phage.

Methods:

Results: Discussion:Friz was found in the soil on the campus of Minnesota State University

Moorhead, south of the main entrance to Langseth Hall at the base of a tree. The presence of a phage was determined by the plaque, or clear spot, in our Microbacterium foliorum plate. The plaque that was used for further testing was very small and clear.

Once the phage was found, it was then isolated and amplified, or reproduced, to gather further information. From our amplification, we deduced that Friz is a very prolific phage and does very well with Microbacterium foliorum. There was no trouble reproducing Friz, and it was a very easy phage to work with. We were easily able to obtain the 10 milliliters of lysate needed for electron microscopy.

Based on the electron microscopy photos of Friz, we determined that Friz is classified as a Siphoviridae. This is based on the long and flexible tail seen in the images. Although it is not certain, based on how clear and defined the plaques that Friz produced, it can be assumed that Friz is a lytic phage. A lytic phage is one that ultimately destroys and kills the host bacteria in its reproductive cycle. We can also determine that Friz is approximately 200 nm long, based on the scale of the electron microscope and the picture shown in figure 4.

Conclusion:

Future Directions:Due to the COVID-19 pandemic, we were not able to sequence the DNA of

our phage as our campus, including our lab, has been closed for the semester. Sequencing our phage's DNA would be the next step in understanding and categorizing Friz. Another direction we would go on would be to test Friz with other bacteria to see how selective our phage is.

References:Ali, Ilzat, et Al. (2018). Phage Discovery Guide. Howard Hughes Medical Institute.Coshell, Ninjata. (09.29.2014). Caudovirales. Wikiwand. Retrieved from https://www.wikiwand.com/en/Caudovirales

Isolation

Purification

Amplification

Electron Microscopy

Siphoviridae

Podoviridae

This research was successful. Our goal for this project was to isolate, purify, and amplify a phage to view under an electron microscope for the purpose of learning and understanding more about phages, and we discovered a very laid back, prolific siphoviridae phage that was easy to work with. We are satisfied with our discovery of new bacteriophage "Friz" that is responsive to Microbacterium Foliorum, and have made breakthroughs on our understanding of the process of phage isolation and other related research.

Myoviridae

Figure 1. The first plate to show signs of a phage. This plate was collected on 01/29/2020. Five very clear, pin head-sized plaques are seen in the Microbacteriumfoliorum which indicates the presence of a phage. One of these plaques was selected for use in the purification process.

Figure 2. The phage was purified using serial dilutions, ranging from an undiluted sample to a dilution of 10^-7. This showed how concentrated and prolific ourphage was. After two rounds of purification, our plaques became very consistent. To start the amplification process, we used a webbed plate to calculate the concentration.

Figure 3. A spot titer plate was created to determine the concentration of the phage to collect a high concentration of a phage solution so that it is easy to find phages in the electron microscope. After three rounds of spot titers, the concentration was high enough titer to use for electron microscopy lysate.

Figure 4. Lysate samples were taken to North Dakota State University to take photos using their electron microscope. Friz turned out to be a siphoviridae phage with a tail of about 200-250 nm and a head diameter of about 50 nm.

Figure 1

Figure 4

Figure 3

Figure 2

Collected soil and snow samples in hopes of finding a phage that reacts to Microbacterium foliorum. Samples were mixed with foliorum and agar to identify phage activity related to the bacteria.

Extracted lysate from concentrated webbed plate of Friz, and used this lysate to capture photos of Friz through an Electron Microscope.

Serial dilutions, flooding past plates, and creating webbed plates continued to raise concentration, to eventually exceed a concentration greater than 5.0 x 10^9, so that it was prolific enough to create a high-titer lysate.

Single out our phage so that no others survive in these specific conditions relevant to Microbacterium foliorumto ensure that we are only working with Friz. This included multiple rounds of filtering and serial dilutions.