Phage on the stage

Abstract

The resurgence of interest in bacteriophages for use in combating antibiotic resistant bacteria is coincident with an urgent call for more effective science education practices, including hands-on learning opportunities. To address this issue, a number of solutions have been proposed, including a large educational experiment, begun in 2007 by the Howard Hughes Medical Institute and currently involving over 85 colleges and universities, which has students discovering unique phages, obtaining images, and purifying phage DNA. A subset of these phage genomes is sequenced and analyzed using bioinformatics tools. Papers describing individual phage discoveries and comparative genomic studies are being published regularly. The vast majority of students in the program are in their first year of college, a critical time in capturing their interest and retaining them as science majors. This viral discovery model is being adopted and modified by a wide variety of educational institutions using a number of different bacterial hosts. In the opinion of the authors, this program and others like it represent a model accessible to virtually any undergraduate setting. And because of these programs, bacteriophage enthusiasts (academics, health professionals, biotechnology companies) can look forward to more well prepared students entering their ranks and should anticipate many more potentially useful phages discovered and characterized.

Keywords:

• Bacteriophages
• bioinformatics
• genomics
• original research
• phage discovery
• phage methods
• undergraduate education

How did an animal physiology professor, a microbiology professor, and a room full of first semester college students uncover a passion for bacteriophage research? Over the course of 2 semesters, this group became very familiar with a multitude of cutting-edge research techniques as the phages began to reveal their secrets. Multiply this scenario dozens of times over, and that's exactly what the Howard Hughes Medical Institute (HHMI) has done with their Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program. This program is based on the PHIRE program (Phage Hunters Integrating Research and Education) at the University of Pittsburgh, where Dr. Graham Hatfull has brought bacteriophage research to high school students as well as first year college students. Through the SEA-PHAGES viral discovery program, thousands of students have participated in bacteriophage research, similar numbers of phages have been discovered and characterized, and hundreds of phage genomes have been sequenced and analyzed by undergraduate students and their faculty.

Several independent elements intersected to set the stage for SEA-PHAGES and other similar programs. Over several decades Dr. Elizabeth Kutter at The Evergreen State College and Dr. Roger Hendricks at University of Pittsburgh pioneered the use of phage biology in undergraduate science education. Dr. Ryland Young's NSF funded program at the Texas A&M Center for Phage Technology (https://cpt.tamu.edu/), implemented and continues to use bacteriophage genomics as a teaching tool. Some 20 years ago, educational researchers began calling for more hands-on, authentic laboratory experiences to improve science education for all undergraduates^1-3 In response to this, numerous research-rich courses were developed, many that were based on genomics of various organisms^4-10 and including international adopters.¹¹ Add to this environment the development of easily accessible tools for bioinformatics and genomic analyses, and the stage was set for an explosion of phage discovery and analyses in the undergraduate science education world.

Participants in the SEA-PHAGES viral discovery program were required to collect copious data on participating students and control groups. The data show that these students stay in college at a higher rate, make better grades than their counterparts who take the same lecture sections with a more traditional lab, and are more likely than their peers to become involved in independent research projects during their college careers.¹² These conclusions are based on students randomly assigned into the program. Further, this is an excellent opportunity to engage minorities, women and first generation college students in STEM careers.^12-15 Students who take a phage course are more confident of themselves as scientists, and are, in their own words, “in love” with science. Based on our several years of experience and discussions with other faculty, we present below what we think are critical components for a successful research course using bacteriophage discovery and analysis:

Every Student Must Be Able to Make His/her Own Discovery

The phage model certainly excels in this category, because of the astronomical number of phage particles predicted on earth.^16,17 So far, approximately 6,750 phages have been isolated by students, 981 have been sequenced, and 466 genomes have been added to GenBank from the SEA-PHAGES program. There is a sense of ownership that comes from the intimate relationship of the students with the biological materials, to the point that they name their own phage and often post the plaque and TEM images on Facebook. This harkens back to Barbara McClintock's “feeling for the organism”¹⁸ in which she intuited mobile DNA elements in the 1940s from her intense personal involvement and detailed observations of maize, her research subject.

There Should Be a Scientific Community of Which the Students Are a Vital Part

Students are involved in the community of their classmates, faculty and TA's, as well as the community of other schools in the phage discovery program and beyond. It is powerful for students at regional and national meetings, such as an annual symposium and American Society for Microbiology meetings, to communicate with other attendees who take their work seriously, showing sincere interest, asking informed questions, and discussing solutions to problems. Being part of an emerging international group with so many other institutions involved is transformative.

Students Must Be Able to Repeat Procedures until They Get Them Right

Learning from one's mistakes and trouble-shooting problems is an important process that builds critical skills. One of the most frustrating things about traditional labs is the severe limitation on time for trial and error, since most traditional labs have a set amount of content that needs to be conveyed to the students. When the experiment fails, the class inevitably just moves on to the next experiment in the schedule. The students then spend inordinate amounts of time writing up why the experiment might have failed, but they never get to address any problems they may have identified. In the phage discovery model, students sometimes have to test 3–4 soil samples before they successfully isolate a phage, then they do repeated rounds of plaque purification, becoming very proficient at pipetting, performing serial dilutions, and calculating titers. After such repetition of techniques, observations, data analysis, and experimental design, students really own the whole process and feel comfortable and confident. This often launches them into very satisfying experiences as teaching assistants or into early opportunities in independent research projects.

Student Discoveries Must Be Significant and Continue to Be Useful and Important after the Students Have Moved on to Other Courses, Individual Projects, and Post-graduate Careers

Seeing their work as part of a real knowledge acquisition process, students understand that even they (typically a college freshman) can make a unique contribution to our understanding of the natural world. When they reach college, students are already of the mindset that a lab is completed in one lab period, and everything is then cleaned up and thrown away. As they participate in archiving their biological products and bioinformatic data, they see that their discoveries are valuable and likely to be studied in the future. Students are amazed when they see names of fellow students on posters and in published papers and realize that they too are doing important work.

So, clearly the students are benefitting. However, they are also making important scientific discoveries. The potential contribution of phage programs to phage biology and genomics cannot be measured or overestimated. Bacterial viruses contain the largest untapped source of unique DNA in the world.¹⁵ There are thousands of potential gene products that have been identified, with no known function. These are being studied using proteomics in addition to wet lab techniques to determine if the predicted genes are expressed and what function these proteins might serve.¹⁹ The large number of phages isolated that infect specific bacteria is providing information relevant to movement of DNA in the environment.²⁰ The burgeoning numbers of new phages discovered and analyzed could help avert the rising crisis in antibiotic resistance.²¹ and be important in food safety and productivity.²² For example, phages that kill Paenibacillus, a serious problem in the honeybee industry, have been found.²³ Unique endolysins that kill bacteria could play a major role in human and animal health and in food safety.²⁴ This list goes on.

The potential for filling the student pipeline leading to life sciences researchers is also significant. Beyond the first year of phage discovery and genomics, many of these students continue to do research on their phages in subsequent years either in small groups or individual projects. These students are already well trained in performing research and they are eager to continue studying their phage or take on other projects. The rare undergraduate who publishes his/her work typically does so in the senior year, but many of our phage hunters are becoming published in their junior, or even sophomore, year. Genome announcements have increasingly become opportunities for making publication feasible for these young scientists.^25-30 Several of these papers were published as a result of a partnership between the Texas A&M program and members of the SEA-PHAGES community. Graduates of the phage discovery program are “hooked” on scientific research in general and are extremely excited about the potential use of phages in medicine, agriculture and biotechnology. Many of them are choosing biomedical research graduate programs, where they hit the ground running. They have the confidence, skills, and experience to begin research immediately, and already have a solid background in bacterial and bacteriophage culture, DNA isolation and manipulation, and genomics.

This may be the first you've heard of such a one-of-a-kind science/education experiment, but you will likely be hearing more. The original host organism used by the SEA-PHAGES project, Mycobacterium smegmatis, has been used for the isolation of 5,909 bacteriophages. Large-scale comparative genomics of these mycobacteriophages have emerged and have been published with dozens of faculty and student authors.^31,32 Faculty involved in the SEA-PHAGES program are developing other host organisms (such as Arthrobacter, Bacillus, Paenibacillus, and Rhodococcus) for bacteriophage isolation in this educational setting and are collecting multiple phages using these hosts. One large and 2 small-scale comparison papers have already been published on the Bacillus phages.^33-35

The program is an amazing success in terms of educational impacts and contributions to phage biology and genomics. The phage discovery model has been implemented successfully at a range of institutions: large public or small private universities, tribal colleges, community colleges, endowed or tuition driven schools, small colleges and high profile ivy league universities. A wide range of students from diverse backgrounds is doing the work and benefitting from the experience: honors students, underrepresented populations, at-risk students, science majors and non-majors.^12-15 As sequencing costs continue to go down and bioinformatics programs become more accessible, such courses are becoming real options for any undergraduate program and even high schools. The cost of a phage discovery lab is now similar to the cost of any other introductory lab, and genomic analysis experiences are virtually free, assuming computer resources. We continue to develop cost saving methods and strategies. Sequencing can be done for under $300 per genome. Electron microscopy costs depend on whether a school has a transmission electron microscope or a relationship with a facility; often members of the consortium cooperate to share these resources.

You may find this model intriguing from an educational as well as a scientific perspective. If so, you can get training (or train yourself), access an extensive array of protocols, and connect with colleagues at neighboring schools. You can easily adapt the educational component into your own scientific areas of interest. If you as a scientist would like to have phages for your favorite bacterium, there is an army of enthusiastic students who could help. And, if you decide to embark on this sort of educational program, there is a community of dedicated faculty willing to share their knowledge and experience with you and your students.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.