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By Julia Ferrante
LEWISBURG, Pa. - Josephine Vargas went to a beach and a compost heap on Long Island in search of her phage. Patrick Reilly explored at a campground in Mifflinburg.
But in the end, Sarah Thibault's soil sample from outside the Biology Building at Bucknell University netted the most compelling example of a phage, or bacteria-eating virus.
"I had no idea what it was going to look like, but when we put it under the microscope we had to take four pictures to get the whole tail and capsid or head," said Thibault, a junior biology major. "It looks like a stereotypical phage, but with a very long tail."
The 12 students in Bucknell's "Phage Hunters" course this past fall were charged with collecting soil samples and identifying phage or viruses that attack bacteria. During the spring semester, the students, mostly sophomores, will analyze the genome sequence or genetic pattern of two of the phage, including the one that shares Thibault's last name.
Taught by biology professors Emily Stowe-Evans and Marie Pizzorno, "Phage Hunters" is part of a national program supported and administered by the Howard Hughes Medical Institute (HHMI) and based on the research of Graham Hatfull, a professor at the University of Pittsburgh. The program, which includes Bucknell and 36 other universities or colleges, provides students with hands-on experience using real research techniques and equipment.
The HHMI program, which began two years ago, supplies most of the materials and training for "Phage Hunters," Pizzorno said. Stowe-Evans learned about the course at an American Society for Microbiology conference, where Hatfull and his students presented a poster about the project.
"The students will come out with more advanced skills that typically they would not get until later in their college career," Stowe-Evans said.
"They also get critical thinking skills," Pizzorno said. "They are doing real research."
Students in "Phage Hunters" gain experience in molecular genetics, electron microscopy and state-of-the-art bioinformatic computer programs, Stowe-Evans said. They also have the opportunity to become a contributing author on a publication about their research and to attend regional microbiology meetings or HHMI seminars.
"I was really excited to take this course because it gives lab experience that can open doors to new research areas in the future, such as phage therapy," Thibault said.
At the start of the course, students were asked to collect soil samples from various locations, Stowe-Evans said. They took the samples to class and tried to isolate phage that specifically infect Mycobacterium smegmatis, a non-pathogenic bacteria related to the bacteria that causes tuberculosis. In most cases, plaques - small clearings - appeared on the plates indicating the presence of a virus or phage. The students repeated the process many times before characterizing the phage and finally traveling to Pennsylvania State University in State College to take pictures through electron microscopy.
The students learned skills such as how to keep a research notebook and analyze data, something biology majors don't usually learn until they are juniors or seniors, Stowe-Evans said.
"Being a part of Phage Hunters has connected the concepts we learned in class and allowed us to actually apply what we learned," said Rochelle Volmerding. "It is a great hands-on experience and is a lot of fun, too."
Benefits of phage
Phage are everywhere - on our skin, in our stomachs and on the ground, but they do not cause diseases in people, Pizzorno said. They are viruses that infect bacteria. Phages are beneficial in aquatic environments and occasionally are used in the treatment of foods such as cheese to prevent bacterial growth.
As phage go through their life cycle, they attach and inject their DNA into cells. The viral genomes then take over cells' machinery so that viral proteins are produced instead of bacterial proteins. Once the progeny virus particles are made, the cells bursts open and the progeny are released to infect new cells, Pizzorno said. Phage progeny can be isolated and become visible through an electron microscope.
"Plaques indicate the absence of cells," Pizzorno said. "We call it a 'zone of death.' As the cells die, you get a clearing in the bacterial growth. Viruses typically have a protein head and a tail. They look like a lunar lander. They attach to the membrane of a bacterial cell and take over, replicate and burst open and go out and look for new cells."
Once the students identified their phage, they chose non-copyrighted names such as LittleThreeC, Bacon and Voldy (a modification of the evil character Voldemort in the "Harry Potter" books).
Jacqueline Bachand, one of the students who traveled to Penn State to photograph the phage under a special microscope, said it was rewarding to see the results of the students' work throughout the semester.
Mapping the genetic code
The class then campaigned to have the DNA of their particular phage analyzed during the spring. Thibault's phage was selected by majority vote. A second phage, found by sophomore Chelsea Dieck and called Little3C, was the runner up and also will be analyzed. The students will look at the raw sequence of the genome and find out if the phages are similar to any previously identified.
"The class will get to analyze the entire genome," she said. "Hopefully, we will have isolated a phage that has never before been seen or studied."
The likelihood that Thibault and Little3C are unique phage is quite high, Stowe-Evans said. Despite the fact that more than 130 phage genomes will be sequenced, no two identical genomes have been identified.
Faria Sanjana said she signed up for "Phage Hunters" because of its connection to the Howard Hughes Medical Institute and other national colleges and universities.
"That really appealed to me, that it was not just Bucknell," she said. "We could talk to other students from other universities about our phage."
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