Biology undergrads land paper in prestigious international journal

“This project meant so much to me, and to be able to make an impact in the field of bacteriophage is a blessing,” said Kim, a former pre-med major who wrote the initial draft of the manuscript and during his senior year put together the figures that drew upon two prior years of research. He, Paul and Diaz edited the manuscript until it was ready for submission in May. An award-winning honors student, Kim graduated summa cum laude five days after the paper published and will enter medical school at Loma Linda University this year. “I hope to conduct research even after I go to Loma Linda School of Medicine to pursue my M.D. I am extremely grateful for Dr. Diaz, my lab members, and the biology department for enabling me to do this research,” he said.

For Paul, also a pre-med major who joined Diaz’s lab in 2020 as a teenage volunteer assistant, the experience confirmed her long-standing interests in scientific research which she plans to incorporate into a future medical career. “Especially because of the research that we've been doing, I realized that the lab is one of my happy places,” she said. “I think what I realized about research is [that] it's almost therapeutic in a way because it allows you to really be able to focus in on something that you know matters.”

For Diaz, an associate biology professor, the publication of his young students’ work in such a prestigious journal was a high point not commonly experienced among the ranks of most undergraduate college students. “I was incredibly excited and proud of Uylae and Elizabeth for getting their work published in a high impact journal, which is something that undergrads rarely get to do, let alone without the help of graduate students or postdoctoral fellows,” he said.

The molecular sciences journal article details findings on the analysis of three bacteriophages, also known as phages. A bacteriophage is a virus that infects and kills bacteria. Phages attack only bacteria and are not harmful to humans. They are simply composed and are abundant throughout Earth’s biosphere in places where bacteria are plentiful such as in soil and water. Because of this and due to their important role in foundational biological concepts such as the definition of a gene and the discovery of messenger RNA as well as their broad applicability to human life, the molecular sciences journal dedicated a special edition to their study, stated journal editors in a description of the special edition.

“… as natural enemies of bacteria, phages are an effective alternative to antibiotic-based treatments of bacterial infections,” the journal editors wrote. “Nowadays, phages are used in food preservation and in decontamination processes,” as well as serve key roles in biological processes.

The three phages at the heart of Kim and Paul’s work, YuuY, KaiHaiDragon, and OneinaGillian, were discovered respectively by Kim and by two former students of Diaz’s SEA-PHAGES class. The students isolated, named, and sequenced the phages which they found in soil samples taken from the local area.

The SEA-PHAGES course was launched in 2017 after La Sierra’s biology department, through Diaz’s initiative, was accepted into the Science Education Alliance administered by the Howard Hughes Medical Institute. It is designed to replace introductory biology classes for high-performing freshmen who have an interest in hands-on scientific research. Students who complete the two-quarter SEA-PHAGES course move to its sequel SEA-GENES which guides students in investigating gene function.

Hunting for solutions

Paul and Kim were able to conduct comparative analysis of the three phages since they were discovered in soil samples from the same area. “It’s really interesting to see how different they can all be,” Paul said.

Kim started the project in 2019 by isolating the phage named YuuY from the soil sample. The name he chose for his phage was inspired by his own life experiences.

“The lesson I learned is that sometimes to get to who and where I am today (uY), it required me to go backwards first (Yu) in order to fix and perfect previous steps,” Kim said. “My mistakes were no longer setbacks, but opportunities to make me a better scientist.”

Kim devised research protocols based on previous papers and the work of other phage scientists. And he annotated the entire YuuY genome himself, normally a group undertaking.

The following year the project expanded to include the other two phages. In 2021 characterization testing continued. That year Kim also started the manuscript detailing his and Paul’s work for submission to the molecular sciences journal which occurred in May 2022.

The overall project goals included assessing whether the three phages would be suitable for phage therapy, a process which uses viruses to treat bacterial infections, and to publish findings in a peer reviewed journal. Experiments were conducted to determine the phages’ reactions to different temperatures and the range of host viruses it could infect, among other things.

Back in 2019 when he started the project Kim estimated a one-year timeline for completion. It took three years and he plans to continue the work by characterizing and analyzing additional phages toward finding therapies in combatting viruses. His lab and other SEA-PHAGES research at La Sierra will continue the work, he said.

Paul joined Kim’s project in 2020. “I really focused on thermal stability assays so can we see at what temperatures these phages survive and the temperature ranges of these phage survivor,” she said.

The Microbacterium, or bacterial strain was coated onto PYCa plate and subsequently spotted with phage to test infectivity. Over 24 to 48 hours the researchers watched for clear patches or holes called plaques to appear in the bacteria indicating that the phage virus was destroying the bacteria. A heating block was used to test phage activity at different temperatures. Similar tests were conducted at differing pH levels. pH is the acidity level which was adjusted with different chemicals to see if the phage treatment would be viable in various parts of the body, such as in the stomach which tends to be more acidic than other parts of the body.

In the end, the young scientists discovered that phage YuuY possessed a broad host range and greater stability at various pH levels and temperatures making it a more suitable candidate for phage therapy to combat bacteria than phages KaiHaiDragon and OneinaGillian.

The mentoring factor

The authors of a paper published in 2011 cited the importance of integrating teaching and research in undergraduate biology education. “Both the National Academies and the American Association for the Advancement of Science recently emphasized that undergraduate education could be improved by a higher level of student participation in authentic research. Their recommendations are of two types: early student engagement in research labs, and enrollment in “research-based” courses modeled on real-world scientific practice,” they wrote.

Students at La Sierra University in general benefit from a low student-to-teacher ratio which facilitates close collaboration with faculty members. In the biology department’s labs including Diaz’s virology lab, the combination of teaching and mentorship involves undergrads in complex projects and field work has resulted in high numbers of them involved as authors in top-tier science journals.

Diaz estimates that between the SEA-PHAGES and SEA-GENES programs more than 50 undergraduates have been co-authors in five publications.

“Dr. Diaz has been like one of the greatest research mentors, and we've been super lucky to have him on campus,” Paul said. “He's taught us a lot of these protocols, he's always been there every time that we've needed anything. He gives us a lot of freedom to figure out what we want to do.”

Diaz likens himself to a basketball coach in the lab. “I help train the students in the various techniques they'll need to develop their projects. We meet weekly to go over the progress they've made and plan the experiments for the following week, and in general [I] provide advice and encouragement throughout the project. It is then up to the students to go out and execute the game plan,” he said.

He also noted that his approach to teaching in the classroom has evolved over time in order to reach current groups of students. “Over the last few years it became clear that learning biology requires integrating new information with the students’ own knowledge and experiences, and delivery of information by itself doesn’t help students develop their own understanding of the material,” Diaz said. “Students have to take an active role in the learning process.”

Kim and Paul’s collaboration began when as a plucky high school senior attending Riverside STEM Academy, Paul emailed Diaz asking whether she could volunteer in his lab. Both Paul’s mother and aunt are alumni of La Sierra University and appreciated their experiences at the school so much so that due to their influence Paul applied only to La Sierra University when considering colleges. At the time, Paul served as a Science Olympiad Coach for the STEM academy and was teaching middle schoolers about microbes.

“I had realized that I wanted to go into the medical field. But then I also realized I had this deep love and passion for biology that had to do with microbes,” she said.

Paul sat for an interview with Diaz before being allowed to work in the virology lab. “Elizabeth had clearly done her homework and came prepared to our interview with questions from the papers she read,” Diaz said. “I was impressed by her grasp of the material even though she was still a senior in high school and due to her desire to do research I paired her with Uylae to work on his project.”

“[Uylae] was one of the greatest mentors when it came to just learning about all of this,” Paul said. “When I first started, I had no idea of how a lab worked. A lot of it was me learning how to use equipment, learning how to use a pipette and over time, Uylae started teaching me protocols for the different experiments that he was doing with his own phage.”

In addition to her work on phages with Kim, Paul has been analyzing the Maize chlorotic mottle virus which is spread by beetles and infects, deforms and kills corn plants. She is building off of preliminary research conducted by other La Sierra biology students.

“I’m working on this virus because it's been so devastating to a lot of crops in Sub Saharan Africa,” she said. “And so by understanding the replication process, we can really open up our field of knowledge and expand on that so that we have more resources to really combat this virus and its replication area.”

No matter the frustrations and rigors of scientific research – it took more than two dozen attempts to get the phage experiments to work --- Paul knows it is meant to be a part of her career path.

“I know there's ways to incorporate research into your medical school training and career and that's something that I definitely know I want to do,” she says. “So that's my goal, to really figure out how to combine research and medicine in the way that my interests and future see fit.”