Marcie the Bacteriophage: Hannah Street, Hunter Stoltz Biochemistry and Biotechnology, Minnesota State University Moorhead, 1104 7th Avenue South, Moorhead, MN 56563 Introduction: Bacteriophage or phage for short is a virus that infects bacteria. To infect bacteria a phage injects its DNA into the bacteria. There are two common phage life cycles. A lysogenic cycle is when a phage injects it genome into the bacteria and it joins with the bacteria’s genome, and a lytic cycle is when a phage injects its genome into the bacteria and creates new phages and then lyses or bursts the cell. A phage consists of a head, tail, and tail fibers. The head of a phage contains DNA or RNA and is enclosed in a protein coat. Siphoviridae is a group of phages with long, flexible, and non-contractile tails. Myoviridae is a group of phages with contractile tails. Podoviridae is a group of phages with short non- contractile tails. Phages are important to learn and study in the world of medicine specifically against multidrug-resistant bacterial infections. Phage therapy may be used as an alternative or supplement to antibiotic treatments (Lim, 2017). We still know so little about phages and every bit of research helps. Our objective was to isolate, purify, and amplify a phage that infects Microbacterium foliorum. Acknowledgments: We would like to thank the Howard Hughes Medical Institute and the Hatfull Lab at the University of Pittsburgh for providing bacteria, support, and training materials. Methods: Purification: ❖ Picking a Plaque: Retrieve phage particles from a plaque to create a lysate. ❖ Serial Dilution: Using serial dilution to the 8th step to create a High Titer Lysate. ❖ Spot Titer: Using Lysate created create a spot titer to determine the concentration of the produced lysate. Results: Isolation: ❖ Collect an environmental sample ❖ Direct Isolation: Use a liquid media to extract phages from the environmental sample. ❖ Plaque Assay: Plate the mixture of top agar, bacteria, and sample to detect the presence of phages on the bacterial lawn. ❖ Picking a Plaque: Retrieve phage particles from a plaque to create a lysate. Amplification: ❖ Spot Titer: Using spot titer plate to determine concentration of lysate. ❖ Webbed Plates: Once concentration is known to be above 1x10^9 pfu/mL or around create webbed plates to produce further high titer lysates. ❖ Flood Plate: Flood the webbed plate with phage buffer to produce a high titer lysate. Figure 1. This was the first plate with phage on it. Circles are marking air bubbles. Figure 2. This is an undiluted plate where you can see the plaques clearly. The plaques are a defined circle but there is some cloudiness outside of the circle. Figure 3. This plate was a spot titer test. There was contamination (the lighter colored spots), but we were still able to calculate the titer by counting three plaques on the 10^-8 dilution. We calculated the titer to be 1x10^11 pfu/mL. Figure 4. This was an electron microscope picture taken with a scale of 0.5 μm. This picture shows multiple phages. Figure 5. This is an electron microscope picture taken with a scale of 100 nm. This shows a close up of Marcie. We estimate the tail to be 170 nm and the overall size of Marcie is estimated to be 210 nm. Sources: Lin D, Koskella B, Lin H. 2017. Phage therapy: An alternative to antibiotics in the age of multi-drug resistance. World Journal of Gastrointestinal Pharmacology and Therapeutics 8(3): 162-173. Microscopy: ❖ Collect High Titer Lysate: From webbed plates produced collect high titer lysate for electron microscope imaging. ❖ Electron Microscope: Phage lysate was collected and given to lab to give us our Electron microscope images of phage. Discussion: Marcie was found in a soil sample near First Silver Lake in Minnesota (46.30913, -95.738483). This sample was collected by our professor. When first discovered, the plaques were small but easily visible. The plaques morphology was somewhat cloudy. This could mean that Marcie is lysogenic. We calculated our concentration by performing spot titer tests. Most of our lysates we collected were considered to be high titer lysate because it was greater then 5x10^9 pfu/mL. We calculated one titer to be 1x10^6 but this was an educated guess because after the 10^-3 dilution the plaques suddenly stopped. We think there was an error made when performing our serial dilutions. We had issues with contamination. At first we believed there was contamination in the bacterial stock, but contamination still occurred after the bacterial stock was replaced. We are still unsure why contamination was happening because it only affected high titer plates. We had electron microscope pictures taken of Marcie at NDSU. These pictures allowed us to see Marcie. Marcie’s tail is estimated to be 170 nm and the overall size to be 210 nm. We determined that Marcie was a siphoviridae because of the long flexible tail. In conclusion, Marcie was a successful phage that survived throughout our research. Marcie was entered into the phage database along with other phages that have been discovered. Future Plans: Due to unforeseen circumstances our research had to be cut short. Future plans for research involving Marcie include DNA extraction and DNA characterization. Furthermore research into what bacteria Marcie is able to attack will be conducted. Using bacteria that is antibacterial resistant Marcie might be able to combat the bacteria that is resistant to antibiotics.