Course-­‐Based Research Grant Proposal -­‐ Best Abstract The course proposed is a full redesign of an existing general microbiology laboratory for undergraduate biology majors, the laboratory component of BIOL 301 General Microbiology. Typically, junior and senior level students take the course, though the student population may begin to include sophomores as the new biology core curriculum is implemented. The redesign will incorporate an authentic research project that spans the entirety of the semester. The research will focus on characterizing microbial populations of fecal indicator bacteria (FIB) in the Macatawa Watershed; the levels of FIB are used by local, state and federal agencies to make decisions about the safety and accessibility of recreational waters. The research will give students the opportunity to consider open scientific questions with relevance to the local community, learn standard microbiological concepts and techniques recommended by the American Society of Microbiology (ASM) guidelines for undergraduate microbiology courses, and introduce modern microbial genomics and comparative analyses into the course. Research Description Escherichia coli and Enterococcus sp. are bacterial taxa widely used in water quality monitoring protocols by county, state and federal agencies. The levels of FIB in recreational waters are used as indicators for decisions about public access to waters with beach closures being a common occurrence in various watersheds throughout West Michigan. Transient, large spikes in the numbers of E. coli have plagued the Macatawa Watershed over the last decade, leading to frequent beach closures in Lake Macatawa and generally lending weight to the negative public view of the water quality in Lake Macatawa. Efforts to study sources of the high levels of E. coli in Lake Macatawa have failed to identify a particular point source, leading to the hypothesis that the majority of the E. coli found in the watershed through routine monitoring is endemic to the region. That is, the populations of E. coli live in the watershed environment year round. Traditionally, E. coli has been thought of as residing primarily in the animal gut. If found in the external environment, the assumption is that the organism has been recently introduced, and thus can indicate the presence of potentially harmful bacteria. If the hypothesis that there are endemic strains of E. coli is correct, there are significant implications for the use of E. coli and potentially other FIB taxa as clear indicators of fecal contamination and health risks in recreational waters. The goal of the research project in the microbiology laboratory is to address this and related hypotheses about the origin and nature of the E. coli found in the local watershed. The general approach to address the hypothesis will be to isolate FIB (E. coli and Enterococcus) from various locations in the Macatawa Watershed. Standard Environmental Protection Agency (EPA) protocols for FIB monitoring will be used [1, 2]. E. coli isolated through the EPA protocols will be purified to homogeneity, yielding pure cultures of each isolate, and the isolates will be archived as frozen stock cultures to generate a culture bank of environmental E. coli strains. The E. coli isolates will then be characterized through a variety of standard microbiological experiments and comparative genomics experiments to test for adaptation to environments outside of an animal gut and pinpoint the origin of the E. coli isolates. Basic physiological characterization of isolates will include ability to form biofilms, resistance to solar radiation, ability to survive starvation conditions, and ability to 1 Course-­‐Based Research Grant Proposal -­‐ Best metabolize at temperatures well below that of the typical mammalian gut. Further characterization would yield profiles for antibiotic resistances found in the environmental strains. In parallel, standard laboratory strains of E. coli originally derived from clinical sources will be characterized in the same experiments as representatives of gut adapted isolates. These experiments will be designed and executed by students, with guidance from the instructor. As wet lab characterization of the strains occurs through the semester, genomic DNA will be isolated from environmental isolates for full genome sequencing using Hope College’s Ion Torrent next generation sequencing platform. This will allow for comparative genomics analyses of the E. coli watershed isolates with the ~2200 E. coli strains that have publically available genome sequences. The comparative genomics experiments will allow for highly specific identification of the watershed strains to help determine origin – we will be able to distinguish strains that have recently been deposited in the watershed versus those that have become endemic to the environment. The strategy will be to identify genes in the genome that are differentially represented between environmental and clinical isolates. Further, we will be able to identify the presence or absence of certain genes known to be involved with causing disease in humans. Results of the research performed in the laboratory will be disseminated through student posters at the Hope College Annual Celebration of Undergraduate Research and Creative Performance, held in the spring of each year. The work will form the basis for subsequent experimentation in my research laboratory, leading to peer-­‐reviewed publication quality data and dissemination. I anticipate that students interested in pursuing the project after the course will appear as co-­‐authors on this type of publication. Further, these students would be good candidates for presenting the work at national scientific meetings, such as the ASM General Meeting held each year in May. Education The incorporation of this project into the standard microbiology lab enables us to address skills and competencies learning goals outlined by the ASM curricular guidelines [3] through an authentic research experience rather than through a series of unconnected, individual exercises. The skills and competencies outlined by ASM in 2010 are quoted below: Scientific Thinking • Ability to apply the process of science o Demonstrate an ability to formulate hypotheses and design experiments based on the scientific method o Analyze and interpret results from a variety of microbiological methods and apply these methods to analogous situations. • Ability to use quantitative reasoning o Use mathematical reasoning and graphing skills to solve problems in microbiology. • Ability to communicate and collaborate with other disciplines o Effectively communicate fundamental concepts of microbiology in written and oral format. o Identify credible scientific sources and interpret and evaluate the information therein. • Ability to understand the relationship between science and society 2 Course-­‐Based Research Grant Proposal -­‐ Best o Identify and discuss ethical issues in microbiology. Microbiology Laboratory Skills • Properly prepare and view specimens for examination using microscopy (bright field and, if possible, phase contrast). • Use pure culture and selective techniques to enrich for and isolate microorganisms. • Use appropriate methods to identify microorganisms (media-­‐based, molecular and serological). • Estimate the number of microorganisms in a sample (using, for example, direct count, viable plate count, and spectrophotometric methods). • Use appropriate microbiological and molecular lab equipment and methods. • Practice safe microbiology, using appropriate protective and emergency procedures. • Document and report on experimental protocols, results and conclusions. Each of these is addressed explicitly through the research project proposed. Certain skills, such as microscopy and media-­‐based identification, will be expanded upon through more traditional laboratory exercises that are interspersed throughout the semester. Additional skills introduced by the research project include analysis and interpretation of genome sequencing data for microbial genomes. Assessment of student attitudes toward science and research will be assessed through CURE surveys, as is standard for CRE programs. Student learning gains will be assessed through pre and post testing of the skills and competencies outlined above, with specific emphasis on a grouping of the learning goals into a data interpretation set – these would include experimental design, evaluation of microbial enumeration data, and identification of microbes. The testing would also include concepts related to microbial genomics – identification of strains, characteristics of genomes, and genetic content of microbes. The research project is designed to engage students in authentic research (hypothesis testing with unknown outcomes) while at the same time ensuring student learning of skills that would be found in any introductory laboratory. Further, the project is designed to extend the set of typical microbiology skills to include more modern, genomics-­‐based approaches to understanding microbes. Rather than limiting project work to a particular set of weeks during the semester, the entire laboratory is designed around the research project. This gives continuity to the research experience and sets up learning of all skills in the context of the project, even if certain skills are not used directly in the project. Students will be reading background literature on the research project, focusing primarily on two recent papers in this area of inquiry [4–6]. With this background, students will discuss the project as a class, followed by experimental design and hypothesis generation within student groups that is expanded to class-­‐wide design. Each student group will be responsible for up to four isolates (essentially 1 per person), and experiments will be conducted as groups. Data from all groups will be compiled as class-­‐wide data sets for comparison and interpretation by each group with their group data and data from the literature. Genome sequencing will occur “offline” (outside of the class) due to the technical nature of the procedures, but concepts will be covered in the class, and students will 3 Course-­‐Based Research Grant Proposal -­‐ Best conduct comparative genome data analyses. Student groups will keep research grade laboratory notebooks. Student groups will present their findings as oral presentations at the end of the semester describing their results and conclusions. Class-­‐wide discussion of group results will lead to overall discussion and conclusions of the project status. Students will have the opportunity to continue the research project during the spring semester in my laboratory. The research project goals require long term, longitudinal analysis of isolates from the watershed – each sampling event represents a snapshot of the watershed. The types of questions being asked demand an answer that can be generalized. Do we see the same strains each year? How do the strains change over the course of the seasons? Are the same results seen in different locations in the watershed? The project lends itself well to implementation in a classroom setting that occurs annually over many years, while providing opportunities for students to continue investigations after the course has finished. Resources The existing schedule for the General Microbiology course will be maintained, requiring one instructor and a student TA. The laboratory meets 4 hours per week during the fall semester. The laboratory is restricted due to biosafety guidelines. The materials include standard microbiology lab materials (that is, running the lab without this project would use very similar types and quantities of materials). There is a dedicated microbiology student preparation crew that prepares most materials. The instructor coordinates the prep crew. Additional costs come in the form of reagents for genome sequencing of watershed bacterial isolates. The estimated cost is $250 per isolate genome sequence. I anticipate that one genome per group would be isolated (typically 6 groups in the laboratory), and up to 10 genomes could be sequenced in one semester. Actual cost for sequencing reagents would be approximately $5,000, with half of that cost dedicated to the microbiology course, itself. The reason for the higher cost is due to the quantities in which the reagents are sold. The amount of time required to execute a laboratory of this type is significantly higher than that of a standard introductory microbiology lab. This includes increased contact time outside of the 4 designated hours and preparation time. Sustainability relies on the ability to obtain sufficient funding for sequencing the isolate genomes. At some level, the cost associated with this part of the course is a function of the modernization of education practices to reflect technologies and approaches being used in 21st Century Biology. Hope College has institutionalized another course (the first year phage course) that also relies on sequencing technology. I obtained an Ion Torrent next generation sequencing platform through the NSF MRI program in 2012 to enable continuation of the phage course genome sequencing at a relatively low cost, and this instrument makes it possible to even propose the type of lab described here. Further, the project described in here provides microbial genome sequences that could be incorporated into the upper level biology course in Bioinformatics, spreading the cost of sequencing across two courses. The Bioinformatics course would use the sequences produced in previous iterations of the microbiology course to perform more sophisticated, in depth analyses of genomes. The data set of available genomes would increase over time. 4 Course-­‐Based Research Grant Proposal -­‐ Best Implementation Timeline Fall 2013 – Initial pilot of the physiological characterization of watershed isolates was performed. Genome sequencing of 6 isolates occurred outside of class after the semester ended. Three students from the class are continuing experiments and preparing a manuscript describing experimental results during the Spring of 2014. Summer 2014 – Development of formal laboratory manual materials for wet lab and development of materials for genome analysis. Prepare pre/post test for assessment of learning gains. Fall 2014 – Implement redesigned laboratory that includes both physiology and genome based experiments. Spring/Summer 2015 – Evaluate initial outcomes and revise for second iteration in the Fall of 2015. Bibliography 1. Method 1600: Enterococci in Water by Membrane Filtration Using membrane-­‐
Enterococcus Indoxyl-­‐B-­‐D-­‐Glucoside Agar (mEI). 2002:EPA 821–R–02–022. 2. Method: 1603: Escherichia coli (E. coli) in Water by Membrane Filtration Using Modified membrane-­‐Thermotolerant Escherichia coli Agar (Modified mTEC). 2002:EPA 821–R–02–
023. 3. ASM Recommended Curriculum Guidelines for Undergraduate Microbiology Education. 2012. 4. Luo C, Walk ST, Gordon DM, Feldgarden M, Tiedje JM, Konstantinidis KT: Genome sequencing of environmental Escherichia coli expands understanding of the ecology and speciation of the model bacterial species. Proc. Natl. Acad. Sci. U. S. A. 2011, 108:7200–5. 5. Walk ST, Alm EW, Gordon DM, Ram JL, Toranzos G a, Tiedje JM, Whittam TS: Cryptic lineages of the genus Escherichia. Appl. Environ. Microbiol. 2009, 75:6534–44. 6. Ingle DJ, Clermont O, Skurnik D, Denamur E, Walk ST, Gordon DM: Biofilm formation by and thermal niche and virulence characteristics of Escherichia spp. Appl. Environ. Microbiol. 2011, 77:2695–700. Budget 5