Summer Workshop in Metagenomics: One Week Plus Eight Students Equals Gigabases of Cloned DNA †

Summer Workshop in Metagenomics: One Week Plus Eight Students Equals Gigabases of Cloned DNA †

Carlos Rios-Velazquez 1,*, Lynn L. Williamson 2, Karen A. Cloud-Hansen 2, Heather K. Allen 3, Mathew D. McMahon 2, Zakee L. Sabree 4, Justin J. Donato 5, Jo Handelsman 6

1 Biology Department, University of Puerto Rico – Mayagüez, Mayagüez, Puerto Rico, 00681
2 Department of Bacteriology, University of Wisconsin – Madison, Madison, WI, 53706
3 Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, Ames, IA, 50010
4 Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT, 06516
5 Department of Chemistry, University of St. Thomas, Saint Paul, MN, 55105
6 Department of Molecular, Cellular, and Developmental Biology, Yale University New Haven, CT, 06511

We designed a week-long laboratory workshop in metagenomics for a cohort of undergraduate student researchers. During this course, students learned and utilized molecular biology and microbiology techniques to construct a metagenomic library from Puerto Rican soil. Pre-and postworkshop assessments indicated student learning gains in technical knowledge, skills, and confidence in a research environment. Postworkshop construction of additional libraries demonstrated retention of research techniques by the students.

INTRODUCTION

Many programs have been implemented to introduce undergraduates to scientific research (10, 8, 4) . At traditionally research-oriented universities, these programs typically involve placing single students in host research laboratories within an overarching program. This can be intimidating to undergraduates who find themselves surrounded by graduate students and postdoctoral fellows with far more experience. Studies point to the need to form a community of peers and mentors to foster a successful undergraduate research experience (1, 18) . A number of successful programs have taken a team approach to utilize undergraduates in research (reviewed in (11) ). In the Joint Genome Institute’s Microbial Genome Annotation Program, undergraduates participate in the annotation of real microbial genomes as the genomes are prepared for publication. Through their participation, students save time that researchers would otherwise devote to annotating each microbial genome. At the same time, these students are also learning how science is done and becoming acquainted with the software analysis tools necessary to carry out research projects in molecular genetics.

Our goal was to design a program with these features using metagenomics as the scientific content. Thus, we designed a week-long immersion workshop in metagenomics, or “library school,” at the University of Wisconsin – Madison for undergraduate students from two campuses of the University of Puerto Rico, and assessed its effectiveness by measuring student learning gains. Library school is another example of how a large research project can incorporate undergraduate research teams. The work performed by student teams increased the number of sites studied and metagenomic libraries built in our laboratories, thereby increasing the likelihood for detecting new genes or molecules. Simultaneously, undergraduates gain knowledge, skills and confidence (16) while learning how research projects involve multiple specialists and produce a wide array of results and impacts.

Library school provided students with intensive training in planning and executing a metagenomic study. Metagenomic analysis provides access to the diversity of enzymes and bioactive small molecules in a microbial community, including those from cultured and uncultured bacteria. Many bacterial species have not been cultured and are recalcitrant to culturing under standard laboratory conditions; it is estimated that less than 1% of environmental bacteria are culturable (reviewed in (17) ). Metagenomics entails extraction of total DNA from a community; so, theoretically, genomes of all organisms in the community are represented. The DNA is cloned into an organism that can be cultured to create metagenomic libraries. Functional metagenomic analysis involves identifying clones that express activities encoded by the metagenomic DNA. In one week, the library school students generated a metagenomic library and performed a selection with a previously generated metagenomic library from Puerto Rico. Working as teams, students isolated soil DNA and built libraries, or pools of Escherichia coli containing soil DNA. Students practiced laboratory skills, discussed the techniques with experts, and synthesized their findings in an oral presentation. In addition to the laboratory practices to which the students were exposed, they also had direct interactions with academic scientists and experienced life at a research-oriented university. A major portion of this experience was to give the students exposure to life as a researcher. Student learning was assessed via pre/post tests, a skills assessment, and an oral presentation rubric. Students later constructed additional libraries in Puerto Rico, indicating retention of technical skills.

METHOD

Approach

The following elements were included in the design and implementation of library school to meet our two goals: 1) to provide an engaging and educational research experience, and 2) to generate metagenomic libraries.

  1. Diverse activities enabled students to practice and gradually learn research techniques ( Table 1). Mini-lectures, laboratory experiments, group discussion, and presentation preparation provided multiple venues for students to engage with new techniques and information.

  2. Continuity of research before, during and after library school facilitated student ownership of projects. Although undergraduate students had been involved previously in all aspects of metagenomics in our laboratories (2, 3) , never has a single student participated from start to finish. Library school was structured to allow the students to see how each experiment fit into the process of building and screening libraries. Library school was part of a larger training program, which has among its goals cultivating scientific resources in Puerto Rico that include both the metagenomic libraries and the next generation of scientists poised to characterize the novel functions they contain.

  3. Assessment of student knowledge, skills, and attitudes throughout. Initial workshop design was based on the six-year training history of the lead instructor. Lynn Williamson had trained 66 graduate students, postdoctoral fellows, and visiting scientists in metagenomic library-building prior to library school. At the end of each day, a roundtable discussion for questions and answers was held, as well as a preview of the procedure to be followed the next day. This activity provided informal feedback to the instructors, and provided the students with an additional opportunity to process the new information and techniques.

  4. Exposure to emerging science disciplines. Metagenomics is a relatively new field of study, but has become the focus of national initiatives (12, 19) and has been the focus for educational initiatives in emerging science (7) .

  5. Strong mentorship. Four trainers, including one postdoctoral fellow, two graduate students, and one research scientist, comprised the mentoring team that worked with the students in the laboratory. Two professors, one from the University of Puerto Rico – Mayagüez and the other from the University of Wisconsin – Madison, also helped design and present library school.

  6. A team approach. The goal of the workshop was to provide an atmosphere where students would work together, ask questions and help each other master new techniques.

Utilizing the framework above, an intensive, hands-on course was designed and evaluated.

TABLE 1   Library school schedule.

 

The participants

The participants in library school were undergraduate students from the University of Puerto Rico – Mayagüez (UPR-M) and – Humacao (UPR-H) campuses. The students’ academic majors included: General Biology, Microbiology, Industrial Biotechnology, Industrial Microbiology, and Agricultural Sciences. The students were part of the Geomicrobiological and Metagenomic Studies of Puerto Rican Soils (GeMS) Project. GeMS has three main objectives: 1) to expand students’ career opportunities; 2) to generate and screen metagenomic libraries from forest soils in Puerto Rico; and 3) to develop a team of young scientists that values the importance of natural resources conservation and reconciles it with emerging technologies.

Prior to attending library school at the University of Wisconsin – Madison, GeMS students participated in workshops and seminars on soil science, ecology, metagenomics, and geomicrobiology. A total of eight juniors and sophomores (five from UPR-M and three from UPR-H), all of whom attended the previous workshops in Puerto Rico, participated in library school. We formed three working teams comprised of two or three students from different institutions. Additional participants included a high school student from Madison, WI and an undergraduate Biology student from Florida A&M University, who attended some of the sessions.

Library school

Library school consisted of approximately eight hours per day of group activities including several short lectures, demonstrations, and hands-on experimental work (see Table 1). Students applied selections to pre-existing libraries to identify antibiotic-resistant clones and prepared their presentations about the research done and concepts learned in library school during breaks. The topics covered during the course included: an introduction to metagenomics, the importance and applications of metagenomics, direct DNA extraction using mechanical methods, large-insert library construction using fosmids, library screening and selection, and vector preparation for small-insert metagenomic libraries (see Table 1).

Course materials and sampling sites

A detailed technical manual was prepared by the trainers based on previous library-building workshops.

Two samples used were from the Caribbean National Rain Forest El Yunque and two were from the Guanica Dry Forest; both sites are UNESCO International Biosphere Reserves. The metagenomic library used to perform the antibiotic selection experiment came from the Tropical Hypersaline Microbial Mats at the Cabo Rojo Salterns in Puerto Rico. The samples were collected and transported to UW–Madison after obtaining the required documentation and permits.

Assessment of student learning and skills proficiency

Several strategies were used to assess the teaching–learning process during library school. A pre/post test, which consisted of ten multiple-choice questions of five alternatives each, in which one of the answers was “I do not know the answer,” was administered to the students at the beginning and conclusion of library school (Supplemental Materials). The questions focused on concepts related to the methods to be covered and performed during the course. A student skill and knowledge self-assessment assessed the participants’ perception of their levels of acquisition of knowledge and proficiency in 20 different techniques related to metagenomic library construction and analysis (Supplemental Materials). The students completed the questionnaire at the end of library school, and the results were compared with those obtained from the previous administration of the questionnaire following prelibrary school activities in Puerto Rico. Five months after attending library school, students were sent an anonymous, Web-based survey requesting demographic information and self-evaluation of their technical skills, their relationship with their research mentors in library school, and their interest and confidence in attending graduate school (13) .

Assessment of student presentations

At the end of the summer course, each team presented a 10-minute oral presentation of its results with one Power-Point slide and a five-minute question-and-answer session. The rubric used in the assessment of the oral presentations is available in Supplemental Materials. The rubric was given to students a day before the presentations for preparation and self-evaluation. A total of four evaluators assessed the presentations: two of the library school instructors and two faculty members, one from Puerto Rico and one from UW–Madison.

RESULTS

Student knowledge assessment

The students participating in library school had some prior knowledge of the topics and increased their knowledge during the course as indicated by a pretest mean score of 19% and a posttest mean score of 79%. The students increased their knowledge of metagenomics, DNA extraction techniques, and library construction techniques. Concepts requiring further clarification included vector structure and function.

Students’ perception of skills proficiency

A total of 20 skills and techniques are included in the student learning goals in GeMS research activities, of which library school is one part. Students’ perceptions after three months of workshops and seminars, but prior to library school (December 2007), were compared with perceptions after library school (Figs. 1A and 1B). Students’ self-reported skills increased following library school in 15 areas and remained unchanged or reduced in five areas (Fig. 1).

 


 

FIG. 1   Students’ perception of knowledge and skills proficiency before (A) and after (B) library school. Skills specifically addressed during library school are listed in bold type. Students were asked to rate their knowledge and skills in: 1) generation of small insert (<10kbp) metagenomic libraries; 2) generation of large inset (> 20 kbp) metagenomic libraries; 3) direct DNA extraction from soil; 4) indirect DNA extraction from soil ; 5) cloning DNA fragments using the TOPO TA system; 6) cloning DNA fragments via traditional methods; 7) using fosmids to build metagenomic libraries; 8) using BACs to generate metagenomic libraries ; 9) transformation of E. coli by electroporation; 10) transformation of E. coli by chemical methods; 11) restriction endonuclease digestion of DNA; 12) determination of optimal endonuclease concentration to generate small genomic fragments by incomplete digestion; 13) amplification of DNA fragments by PCR; 14) analysis of DNA by standard gel electrophoresis; 15) analysis of DNA by pulse-field gel electrophoresis; 16) detection of activities in metagenomic clones; 17) generation of recombinant DNA libraries from metagenomic libraries; 18) in silico analysis of nucleotide and amino acid sequences; 19) plasmid extraction from transformed E. coli cells; and 20) DNA sequencing. The scale used by the students to assess their level of knowledge or proficiency was high, intermediate, or low.

Most students reported previous knowledge and expertise in: direct DNA extraction (87%), electrophoresis (62%), generation of small size DNA metagenomic libraries, indirect DNA extraction, cloning, transformation, restriction endonuclease digestions, and plasmid extractions (50%).

Following library school, the students reported a higher level of proficiency in 12 skills (1, 3, 4, 6, 7, 9, 10, 12, 14–17; listed in Supplemental Materials), with a change evident among 7% (generation of small-sized metagenomic libraries) to 45%–50% (direct and indirect DNA extraction) of the students. Nine areas (2–4, 7, 8, 14–16, 19) were directly addressed in library school. Students reported intermediate to high knowledge and proficiency in seven skills, and low to intermediate knowledge and proficiency in two areas (Fig. 1).

Final presentation

Each team presented one talk to the course members and mentors. Presentation scores ranged from 78% to 98% of the total points available on the rubric. Student self-evaluations were more critical than those of mentors (Table 2).

TABLE 2   Assessment of oral presentations by students participating in library school.

 

Postlibrary school student surveys

An anonymous, Web-based survey covering impressions of and skills learned during library school was sent to all participants. Six of eight surveys were returned. All respondents expressed confidence in making contributions to research, being able to explain their results to others, and their ability to succeed in a science career (Table 3A). All respondents also identified with their mentor and the other workshop leaders as role models (Table 3B).

TABLE 3A   Student survey results: self-reported confidence in science success.

 

TABLE 3B   Student survey results: majority of students report comfort with research environment.

 

Experimental results from metagenomic library construction

During library school, the students constructed a fosmid library containing approximately 51,200 clones and 1.536 Giga base pairs of DNA from Guanica Dry Forest soil (Fig. 2). The students built four more fosmid libraries in Puerto Rico, one from Tropical Rain Forest soil (4,571 clones) and three more from the Guanica Dry Forest sample (786,748 clones total).

 


 

FIG. 2   Dry forest library characterization. Eighteen clones were chosen randomly to determine the presence of an insert via fosmid DNA purification, restriction endonuclease digestion, and gel electrophoresis. All but one clone contained insert DNA. Two molecular markers were used: 1 kb ladder (Promega Corporation, Madison WI) (lane 1) and Yeast Chromosome PFG Marker (New England BioLabs, Ipswich MA) (lane 2). The arrow indicates where linearized vector runs (approximately 7.5 kbp).

DISCUSSION

Library school schedule

Library school provided participating students with diverse experiences that produced measurable learning gains (Table 1). The students attended mini-lectures, constructed and screened metagenomic libraries, attended presentations about research techniques and graduate school, and participated in recreational activities. The goals of this combination of activities were to bring balance to the learning experience and to allow the students to become familiar with the campus of a large research university. The six-day timeframe was sufficient for the students to build one metagenomic library, but an additional two or three days would be necessary for students to properly characterize and store the library.

Metagenomics as a teaching tool

Constructing and screening metagenomic libraries (CSML) has several characteristics that make it a powerful teaching tool. CSML presents opportunities for both success and trouble-shooting. Protocols are well-established, but need to be optimized to each soil type (6, 9) . CSML involves a variety of techniques, and allows the development of multiple research skills of different complexities. For example, screening libraries involves basic microbiology techniques such as bacterial culturing, and library construction requires molecular techniques including cloning and restriction analysis. In addition, CSML provides students the opportunity to design a screen or selection of the libraries based on the biology of their environmental sample. Designing and executing a functional metagenomic screen requires independent research of the literature, and encourages interactions among students from different disciplines. During library school, students in diverse majors ranging from Agricultural Sciences to Biotechnology collaborated to construct a metagenomic library that they then screened for activities of their choice in Puerto Rico. Finally, CSML exemplifies the interdisciplinary and transdisciplinary nature of science. Students experience first-hand that biochemistry, genetics, and computer science play an essential role in successful library generation and analysis (interdisciplinary). They come to appreciate that their findings may impact research and politics in bioengineering, business administration, ethics, and natural resource conservation (transdisciplinary). As students participated in library school, their perceptions of their own knowledge and skills shifted (Fig. 1), occasionally indicating they felt less knowledgeable about certain skills, especially those not covered in the workshop. Perhaps along with students learning new information, their experiences showed them what they did not know.

Undergraduates as a tool in metagenomics research

A significant bottleneck in functional metagenomics is the low frequency of active clones. For example, successfully identifying clones encoding phenotypes of interest ranges from 1:456 to 1:118,620 in libraries of a Wisconsin agricultural soil, depending on the activities assayed (15, 18) , which is similar to the results from other metagenomic surveys for biocatalysts (Sabree, unpublished analysis). Functional metagenomic screens and selections are labor intensive, but as demonstrated by library school, many of the required techniques are well within the capabilities of undergraduates. Harnessing the potential of undergraduate researchers to perform much of the initial clone characterization is one way to expand the size and scope of functional metagenomic screens and selections (5) .

During the one-week library school, eight students provided the first glimpse into the microbial diversity of two unique habitats, the Tropical Rain Forest el Yunque and the Guanica Dry Forest. They also acquired knowledge and skills as measured by the pre/posttests, skills assessment, and evaluation of oral presentations by the instructors and students. Library school married key features of the classroom and research lab and successfully integrated undergraduates in cutting-edge microbiological research. At the end of library school, the students were asked to describe the experience in one word. They answered: “enriching,” “guiding,” “transforming,” “gratifying,” “new,” “decisive,” and “rewarding.” Once back at their home institutions, the participants were able to continue applying what they learned about functional screening to the generated libraries, as well as performing a diversity study based on sequence analysis. Some examples of the functional analysis involve selections for resistance to different antibiotics including tetracycline, ampicillin and kanamicin, and screening for activities such as proteases, lipases and ureases (data not shown). The students presented their findings at local and national conferences. Library school may serve as a model of actively engaging students in state-of-the-art research; this workshop is being adapted to run with additional cohorts at Yale University.

SUPPLEMENTARY MATERIALS

Appendix 1: Oral Presentation Rubric, Pre/Posttest Questionnaire, and Self-assessment Chart

ACKNOWLEDGMENTS

We thank the Center for Hemispheric Cooperation (CoHemis) at UPR-Mayagüez, Dr. Lilliam Casillas at UPR-Humacao, and the Puerto Rico Department of Natural Resources and the Environment. Dr. Christina Matta assisted with production of the laboratory manual. Data on student learning and perceptions was collected and analyzed with the assistance of Dr. Christine Pfund at the University of Wisconsin-Madison with approval from the Human Subject Institutional Review Board (protocol #SE-2007-0192). The authors declare that there are no conflicts of interest.

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*  Corresponding author. Mailing address: Biology Department, University of Puerto Rico – Mayagüez, Call Box 9000, Mayagüez, Puerto Rico, 00681-9000. Phone: 787-832-4040. Fax: 787-834-3673. E-mail: carlos.rios5@upr.edu .

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† Supplemental material available at http://jmbe.asm.org ( Return to Text )

DOI: 10.1128/jmbe.v12i2.177
Journal of Microbiology & Biology Education , November 2011
Copyright © 2011 American Society for Microbiology . All Rights Reserved



JMBE
ISSN: 1935-7885