The Phage Hunters Advancing Genomics and Evolutionary Science (PHAGES) program from the Howard Hughes Medical Institute’s Science Education Alliance provides a year-long, classroom-based research experience for university freshmen. In PHAGES, students each isolate and characterize a novel bacteriophage, a virus that infects bacteria, as well as participate in the annotation of a phage genome. The University of North Texas implemented PHAGES in the fall of 2009.
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The Howard Hughes Medical Institute (HHMI) created the Science Education Alliance (SEA) and introduced the National Genomics Research Initiative (NGRI) in 2008 with a cohort of 12 institutions. The NGRI program, which was renamed Phage Hunters Advancing Genomics and Evolutionary Science (PHAGES) in 2011, provides a year-long, classroom-based research experience for university freshmen. In PHAGES, students each isolate and characterize a novel bacteriophage, also known as phage, which is a virus that infects bacteria. Each year, the genome of one or more phage is sequenced, and students then identify the location of the genes to create an annotation of the phage genome. The University of North Texas (UNT) was selected to join the SEA in 2009 as a member of the second cohort and received funding from HHMI for materials and supplies for the first three years of the program. The first PHAGES class at UNT began in the fall semester of 2009.
Description of the PHAGES Courses
The experimental procedures and scientific goals of the PHAGES program are provided through the SEA, but each institution determines how they will organize and implement the course. At UNT, the PHAGES program was developed as a two-semester course sequence named Introductory Biology Research Laboratory I and II. A faculty member and a graduate student teaching assistant together teach each section of the laboratory. The first course, offered each fall, focuses on the isolation and characterization of bacteriophage and takes place in a laboratory setting. The students meet twice per week for 3 hours each and receive 2 semester credit hours (SCH) for the course. The second course in the sequence is primarily devoted to analysis of the genome sequence and meets each spring in a computer laboratory classroom. This course meets twice per week for 2 hours each and earns 1 SCH. Students at UNT take each of these courses concurrently with the Principles of Biology I and II course sequence. The Introductory Biology Research Laboratory I and II are used as replacements for the normal laboratory course sequence associated with those lectures. Because of the nature of scientific research, students are expected to be available for some additional time outside of the scheduled laboratory meetings as needed.
In Introductory Biology Research Laboratory I, students begin the semester without many of the basic laboratory skills needed for the project. The instructors provide introductions and demonstrations of these skills on an “as needed” basis. The structure of the program allows students to repeat and practice the primary skills many times, so they quickly become proficient.
There are three scientific goals for the fall course: 1) each student will isolate a unique bacteriophage, 2) an electron micrograph will be obtained for each phage isolate, 3) purified DNA will be obtained for each phage, and 4) a restriction digest will be completed for each DNA sample.
Students begin the semester by collecting soil samples to be used for phage isolation. Two procedures are used, an enrichment method and a direct plating method, to expose a susceptible host to viruses from the soil sample. The host bacterium used by the PHAGES program has been Mycobacterium smegmatis mc2155. This organism is non-pathogenic and suitable for use by freshman scientists, but serves as a good model for isolation of phage that might also infect medically-important mycobacteria such as the causative organisms for tuberculosis or leprosy. Students look for the formation of plaques, which are zones of clearing caused by viral infection and growth, on a bacterial lawn to indicate the presence of phage. Plaques are then selected for purification, a process that requires multiple repeated isolations to be sure that only a single type of virus is present in the sample. Following purification, the student then isolates DNA from the sample and analyzes the DNA through restriction digests. Restriction digest patterns are one method used to assist students in determining if their phage is unique and if the phage might be similar to other previous isolates, as well as may be used to estimate genome size. Phage samples are also prepared for analysis by transmission electron microscopy to determine the structural type and size of the phage particles. All the information about each phage is presented near the end of the semester during a “phage olympics” in order to select the isolate or isolates that will be sent for DNA sequencing.
Introductory Biology Research Laboratory II begins in the spring semester and requires the students to learn a new set of skills. DNA sequencing of the sample from the fall occurs during the semester break and is ready for analysis sometime early in the spring. To analyze this data, students receive instruction and practice using several computer-based tools for bioinformatics and genome analysis. Working individually and in small groups, students identify and describe the elements of the genome in order to produce an annotated genome. The class as a whole reviews the work and comes to a consensus on the location and type of each genetic element. The final annotated genome is then sent to the SEA program and the University of Pittsburgh for quality control. Following review, the annotated genome is submitted to Genbank, a database where the data is published and made available to the scientific community.
Depending on the phage isolate, cluster type of the phage, or unique properties determined during characterization, students may have additional opportunities to contribute to peer-reviewed scientific articles about the isolate or phage biology.
Students in the Program
Different SEA institutions have selected a variety of student populations for the PHAGES program. At UNT, the students recruited and admitted into the program are “typical” freshmen biology and biochemistry majors. No special qualifications are required of students other than a willingness to apply, eligibility to enroll in Principles of Biology, and an understanding that the PHAGES course has both higher workload expectations as well as potential rewards compared to the traditional lab course. Potential participants are recruited through mailings to admitted freshmen, a website, and orientation presentations. For 2009 and 2010, up to 24 students per year were recruited to make one course section. Beginning in 2011, two sections of 16 students each were filled.
A total of 79 students have participated in the fall course over the first three years of the program. Sixty-six of those students completed the full year.
In the first three cohorts, a total of 77 phage isolates have been purified and characterized. One student dropped out of the university prior to completing the semester, and one failed to isolate their own sample so instead assisted another student. For the 77 isolates, all the scientific objectives (DNA sample, restriction digest, and electron micrograph) were achieved.
Four UNT-isolated phages have been sequenced and annotated through the PHAGES program. Mycobacterium phage Adephagia (GenBank Accession #JF704105) and Alice (GenBank Accession #JF704092) were completed by the 2009 cohort, Mycobacterium phage Timshel (GenBank Accession #JF957060) by the 2010 cohort, and Mycobacteriophage ElTiger69 (GenBank Accession #JX042578) by the 2011 cohort.
Course participants have also been encouraged to present their work outside of class. Each year in June, a student representative of the program attends the SEA Symposium in Ashburn, Virginia at the HHMI Janelia Farms Research Campus to present that year’s work to other SEA member institutions. In the spring of 2010, two students presented their work at UNT’s University Scholars Day 2010 (one paper, one poster). To increase the number of students presenting their phage research, the instructors added a poster assignment to the class for the 2010 and subsequent cohorts. This change along with renewed efforts to encourage presentations resulted in six posters presented by PHAGES students at the Texas Branch American Society for Microbiology (ASM) Spring Meeting 2011, as well as seven posters and one paper at the UNT University Scholars Day 2011. In 2012, three PHAGES students gave posters at the Texas Branch ASM Spring Meeting, and six had posters at the UNT University Scholars day.
A significant accomplishment of students from the 2009 cohort was the contribution of 4 UNT PHAGES students as co-authors on a peer-reviewed paper in the high impact journal PLoS One (Pope et al., 2011). This paper discussed characteristics and comparative genomics of Cluster K mycobacteriophage, including Mycobacterium phage Adephagia which was isolated at UNT.
Overall, the implementation of this new research-based laboratory course for freshmen was successful at UNT. Student retention in the course was very good each year. While more faculty time was devoted to this course than to a typical laboratory, several positives were noted by the instructors. These freshmen students quickly moved from being complete novices to being comfortable in the lab in just a few short weeks. The instructors’ also enjoyed seeing the excitement on the students’ faces when they saw the EMs of their phage for the first time. Another positive was seeing students continue to work on obtaining EMs and restriction digests even after the phage to be sequenced had been selected and sent to the sequencing centers.
The addition of the poster assignment appears to have helped UNT to increase the number of NGRI students who were willing to present their work at outside of class opportunities. The 2009 cohort was presented with the same presentation options as later cohorts, but very few were willing to do so. By having the students already working on a poster, there was less additional work needed in order to submit to one of these opportunities. We were pleased with this result and plan to continue this assignment in future semesters.
The PHAGES program offers an extraordinary opportunity for beginning freshmen to gain valuable research experience in their major field. Even though HHMI support for the course has ended, the course will continue to be offered each year since the benefits of the program for UNT students are so great.
- Pope, W.H., Jacobs-Sera, D., Benjamin, R.C., Davis, A.J., DeJong, R.J., Elgin, S.C.R., Ferreira, C.M., Guilfoile, F.R., Forsyth, M.H., Harris, A.D., Harvey, S.E., Hughes, L.E., Hynes, P.M., Jackson, A.S., Jalal, M.D., MacMurray, E.A., Manley, C.M., McDonough, M.J., Mosier, J.L., Oosterbanna, L.J., Rabinowitz, H.S., Rhyan, C.N., Russell, D.A., Saha, M.S., Shaffer, C.D., Simon, S.E., Sims, E.F., Tovar, I.G., Weisser, E.G., Wertz, J.T., Weston-Hafer, K.A., Williamson, K.E., Zhang, B., Cresawn, S.G., Piuri, M., Jacobs, W.R., Jr., Hendrix, R.W., and G.F. Hatfull. 2011. Cluster K mycobacteriophages: Insights into the evolutionary origins of mycobacteriophage TM4. PLoS One 6(10): e26750. doi:10.1371/journal.pone.0026750.