Edited by: Carlos Christopher Goller, North Carolina State University, United States
Reviewed by: Lorelei Elizabeth Patrick, Fort Hays State University, United States; Amanda E. Sorensen, Michigan State University, United States; Anil Kumar Challa, University of Alabama at Birmingham, United States; Adrian Shieh, Drexel University, United States
This article was submitted to Systems Microbiology, a section of the journal Frontiers in Microbiology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Course-based undergraduate research experiences (CUREs) are an effective means of transforming the learning and teaching of science by involving students in the scientific process. The potential importance of the microbiome in shaping both environmental health and disease makes investigations of microbiomes an excellent teaching tool for undergraduate microbiology. Here, we present a CURE based on the microbiome of the bean beetle (
In recent years, course-based undergraduate research experiences (CUREs) have gained widespread attention as being effective alternatives to “cookbook” style teaching approaches in science, technology, engineering and mathematics (STEM) (
Interdisciplinary research on microbiomes, or the collection of microorganisms and their genes within a given environment, has uncovered the vast diversity and complexity of the microbial world. Microorganisms and their activities are essential for maintaining both human and environmental health (
Despite the educational potential for laboratory classrooms, microbiome research can be challenging. Many biological systems are highly complex, involving a diverse array of microbes with confounding variation both within and between populations (
Bean beetles (
The Bean Beetle Microbiome Project is a large-scale, multi-year STEM-Education research collaboration with the overarching goal of understanding the role of student autonomy in using scientific practices in a discovery CURE for students across diverse institutions. Here, we present and describe the course materials developed for the Bean Beetle Microbiome CURE that can serve as an inquiry-based course curriculum for undergraduate microbiology laboratories. We also present preliminary survey data from upper-level undergraduates at three different institutions who participated in the first set of implementations of the BBMP-CURE during the Fall 2019 academic term. In the BBMP-CURE, students integrate microbiological, molecular, and bioinformatic techniques to characterize both culturable and unculturable members of the beetle gut-microbial community. We discuss how the CURE can be implemented in either 6-week (half semester) or 12-week (full semester) versions, allowing flexibility within the curriculum. The 6-week format can also be implemented by instructors within institutions that follow the quarter-system. Depending on the research interests of the faculty, many aspects of the CURE outlined below can be easily modified such that students and instructors can take greater ownership of the research questions asked, methods used, and overall research experience. Student-generated data may contribute to ongoing faculty research that subsequently leads to publications. Additionally, faculty have the option of sharing their classroom data within a curated open-access database on the Bean Beetle Microbiome Project website, facilitating the generation of broader research questions across laboratory classrooms and institutions.
The activities below are designed for students to expand on laboratory skills that they may have practiced in previous introductory laboratory courses (e.g., pipetting, culturing bacteria, and record keeping). However, activities can be easily amended for an introductory class such that basic laboratory skills can be introduced at the time of or prior to performing the activity.
The overall learning objectives for the CURE are for students to be able to:
Describe the impact of microbes on our living planet and their host environments.
Sample and compare microbial communities (microbiomes).
Formulate testable hypotheses to address a research question and design an experiment to test the hypotheses.
Identify and apply the microbiological, molecular, and bioinformatic techniques used to study microbiome data.
Analyze 16S rRNA gene sequence data with common techniques used in microbiome research.
Interpret figures generated from microbiome sequence data.
Communicate findings to peers via oral or poster presentations and scientific writing (report).
To facilitate project ownership, an additional learning objective may be for students to “Discuss and pose a meaningful research question that builds on prior research.” Learning objectives can be modified and amended by the faculty based on the specific research questions asked. Published example rubrics for evaluating student presentations are available (e.g.,
A living culture of bean beetles may be purchased from Carolina Biological Supply Company (Burlington, NC, United States, item number 144180) or Ward’s Science (Rochester, NY, United States, item number 470163-616). Beetles are easily maintained on a countertop in small jars or containers with dried beans (
Maintaining bean beetles in the laboratory classroom. Adult (
While rearing beetles for classroom activities, it is important to plan accordingly to synchronize emergence times with experiments. Beetle cultures should be grown for a few months in consistent laboratory conditions on the same bean species so as to accurately predict emergence times. Additionally, maintaining replicate lines (on the same bean-host) that have alternating emergence periods can ensure a consistent supply of adult beetles. More details on working with bean beetles can be found at
A current list of all equipment, supplies, and reagents used in the different modules of this CURE can be downloaded at
For the bioinformatic analysis portion of the CURE, we designed the activities to utilize free, web-based software that does not require advanced computer science skills (e.g., command-line usage or coding skills). Access to a computer lab would facilitate the implementation of the bioinformatical analysis activities; however, students also may use their personal laptop computers while accessing a university internet connection. Alternatively, bioinformatic analysis may be designated as outside laboratory assignments, allowing students more flexibility to access the technology (computer, internet-connection) necessary to complete the analysis. For example, in Spring 2020, we conducted bioinformatic analyses with undergraduate students in a completely on-line distance learning format.
For taxonomic identification of isolated colonies from beetle gut-homogenates, The Basic Local Alignment Search Tool (BLAST)
The lessons presented here make use of both Sanger and Next-Generation Sequencing technology in order to provide students a greater breadth of understanding of the tools available to study both cultivable and non-cultivable bacteria at the genetic level (single colony/species vs. whole communities). Currently, the cost for 16S V4 Miseq amplicon sequencing ranges from $55 to $85/sample depending on the number of samples in a single run and whether you use a commercial or academic institution sequencing facility. A meaningful microbiome experiment will likely require a minimum of five samples in each of two treatments, or a total of 10 samples, so whole community sequencing will cost between $550 and $850/class. The cost of NGS-technology can be prohibitive (
The CURE outline below was designed for a laboratory course meeting once a week for a minimum of 3 h. The outlined materials and procedures can accommodate a laboratory of 24 students. Activities were designed for students to work as collaborative teams (e.g., 6 groups of 4 students each). Notably, while the BBMP-CURE was originally designed for hands-on laboratory instruction, much of the components, particularly the modules relating to sequence and community analysis, can be performed in fully virtual-classroom formats.
All handouts for each laboratory session can be found at
The BBMP-CURE has the flexibility of being carried out in either full or half-semester implementations. Both full- and half-semester timelines can be found in
The first class period serves as an introduction to microbiome research in general. The introduction includes a discussion of how insect microbiomes can be used as research models. An introduction into bean beetles as the model organism provides information on bean beetle ecology, sex-based identification, life-cycle, and agricultural impact. Additionally, students are asked to read published studies relating to bean beetles and microbiome research in various biological systems. Background reading can be assigned as either as a pre-lab or in-class assignments (see suggested reading materials).
The introductory lecture and reading assignments provides the background information and context to facilitate the development of a research question, hypothesis, and experimental design, including identification of control versus experimental groups. The BBMP-CURE uses a guided-inquiry approach to guide students through these initial stages of the project. The research question to be studied is either provided by the instructor (faculty-driven research question), or it is generated by the students themselves through an iterative process of discussion and feedback. Regardless of how the research question is generated (either by the students or by the instructor), students are expected to predict the outcome of the study (generate a hypothesis), as well as to design and conduct the experiment to test their hypothesis, including identifying the control versus experimental groups. Instructors may choose to structure a more faculty-driven or student-driven approach. The approach taken may depend on student level (e.g., freshman versus senior), course type (e.g., introductory vs. advanced courses), or course objectives. Additionally, the instructor may choose to have students formulate their research question(s) based on class consensus, or alternatively, if the budget permits, different groups may choose to answer different questions. Example research questions that have been generated by students of the BBMP-CURE using a guided-inquiry approach can be found in
By week 2, students and instructors have agreed upon the research question(s) to be investigated. Subsequently, students extract DNA from beetle gut homogenates for whole-community sequencing. The timing of DNA extraction depends on the nature of the experiment being conducted. Comparisons of microbiomes from individuals taken from mass cultures (for example, comparing the microbiomes of males and female beetles) may be performed as early as week 2 or 3 if those cultures were previously established. However, a manipulation experiment will require three weeks or longer for the development and emergence of adults (for example, the effect of a host shift on the microbiome community). In classroom settings, waiting for adult emergence is necessary, as larvae are embedded in beans and may be hard for students to successfully isolate without contamination. The steps outlined below describe DNA extraction using the Qiagen DNeasy Blood and Tissue Kit. Qiagen DNeasy Blood and Tissue kit was chosen for this module based on previous literature that demonstrates its successful retrieval of insect-microbiome DNA (
DNA is extracted using a modified protocol for the purification of total DNA from insects (see both Instructor and Student Handouts on DNA extraction for the full semester implementation
It is not uncommon for students to extract insufficient DNA on their first attempt. Therefore, week 3 of the full-semester implementation has been designated as an iteration day, allowing students the opportunity for a second attempt at DNA extraction. Students can repeat unsuccessful extractions, or students who successfully extracted sufficient DNA in week 2 can use the time to isolate DNA from an individual from another treatment group to increase sample size. Alternatively students who do not need to repeat an extraction can proceed to culture bacteria from gut homogenates (see below).
The DNA extraction step is ideally performed early in the semester (week 3) in order to provide ample time for sequencing to be performed. Although typical turnaround times for paired-end whole community sequencing can vary, we have allotted ∼4 weeks from the time the DNA is sent out to retrieval of sequence data for community analysis.
Once DNA extractions have been performed, students initiate culture-based analysis of microbes isolated from beetle gut-homogenates. Beetles are surface sterilized and transferred to a 1.5 mL microcentrifuge tube containing 500 uL of 0.9% sterile saline solution. Each beetle is crushed with a sterile pestle and cell-homogenates are serially diluted and plated on different solid-media to obtain bacterial cultures from the beetle microbiome. The current module outlines the use of a general growth media (nutrient agar) as well as the use of selective media for Gram-positive (phenyl-ethyl alcohol) and Gram-negative (eosin-methylene blue) bacteria. Students allow their plates to grow at room temperature (25°C) until the following class period. However, the instructors may opt to utilize any number of general or selective medias to appropriately pursue the specific research question being addressed by their students. Additionally, initial growth in liquid cultures can be used to pursue questions related to physiological or metabolic capabilities of the beetle microbiome.
The following class period (week 4), students check the growth of their microbial cultures. They observe and record the phenotypic characteristics of grown colonies. Using a colony-based PCR approach, students then amplify DNA from specific colonies of interest for taxonomic identification via Sanger sequencing of the 16S rRNA gene (see both Instructor and Student Handouts on colony-based PCR). Here it is important to save students’ plates, as the following week will allow for iteration (a second attempt at PCR for any groups who do not obtain a successful amplified product). Additionally, students who took advantage of the iteration for DNA extraction in week 3 may catch-up this week by crushing beetles and plating the gut-homogenates onto solid-media.
In week 5, students who performed colony-based PCR in week 4 visualize their amplified products using gel electrophoresis. Additionally, week 5 can serve as an iteration day for students who may still need to catch up (either they need to perform colony-based PCR on their isolates for the first time, or their PCRs did not yield enough DNA and they need to redo their PCR). All successfully amplified DNA products should then be sent out for Sanger sequencing.
As Sanger-sequencing turn around is typically fast (∼24–48 h), students should have their Sanger-sequence data by the following class period (see Student Handout on BLAST analysis of sequencing data). Students use the NCBI-nBLAST database to taxonomically classify the colony-based PCR sequences, and create a taxonomy table of the bacterial genera identified in the microbiomes of the beetles in their experiment. This class session serves as an introduction to bioinformatics tools and computational software to compare genetic sequences for taxonomic relatedness. The concepts learned in these sessions will continue to be built upon as they proceed through the semester.
In week 7, students are introduced to community analysis. They are first introduced to this concept by performing community analysis on the phenotypic data that they previously collected on microbes isolated on solid-media. They connect the phenotypic data from week 4 with the colony-based PCR sequence data from Sanger sequencing. They calculate alpha-diversity statistics based on the taxonomy table of different taxa identified from the colony-based PCR sequence data and determine similarities and differences in diversity between samples (
By week 8, the whole community (paired-end) sequence data should be available. These data permit students to expand on the community analysis by identifying bacterial taxa in the whole-community dataset using DNA Subway. Weeks 9 through 13 are then reserved for bioinformatic analysis of microbial community sequence data. Since this is the most challenging technical aspect of the CURE, several sessions are allotted to complete this activity. Multiple options are available here to cater to varying degrees of experience and pre-requisite knowledge of students and instructor. These include community analysis using the R Statistical Package, spreadsheets in Excel or Google Sheets, or the free web-based Shiny App ranacapa (
In the final week, students develop their scientific communication skills by presenting their results. Instructors may choose presentation methods to match their course learning objectives (scientific abstract, full scientific report, seminar style presentation, poster presentation). Undergraduates may also take advantage of opportunities to present research outside of the classroom at their home institutions (for example, a departmental undergraduate research symposium or college-wide science symposium).
Similar to the full-semester implementation, the first class period for the half-semester implementation serves as an introduction to insect microbiomes in general and bean beetles as the model organism. Additionally, the research question and experimental design should be discussed and agreed upon during the first class session. However, the introduction period is shortened such that students initiate their research project in the first class period. Once students and instructors agreed on the research question(s) to be investigated, students are provided with their specimen beetles and follow surface sterilization and homogenization steps to plate beetle-gut homogenates onto general and selective media.
The following class period (Week 2), students check the growth of their microbial cultures, observe and record the morphological characteristics of grown colonies, and perform colony-based PCR on their isolated colonies for taxonomic identification via Sanger sequencing (see both Instructor and Student Handouts on colony-based PCR for the half-semester implementation
In week 3, students perform gel-electrophoresis on their amplified DNA (see Instructor handouts on electrophoresis). Typically, electrophoresis can be completed in about 1 h, at which point any groups that did not successfully amplify DNA may elect to re-do their PCR on a picked colony. Then, successfully amplified DNA products may be sent out for Sanger sequencing. In a typical 3-h laboratory course, students would have enough time to run a gel and re-do PCR for any unsuccessful amplifications. However, running a gel on the second PCR attempt to confirm amplification may require students to perform laboratory work outside of scheduled class time.
The following class periods (weeks 4, 5, and 6), students begin the bioinformatic analyses. They use the NCBI-nBLAST database to taxonomically classify sequences (see Student handout on BLAST analysis of sequencing data), perform community analysis on phenotype and colony-based PCR sequence data (see student handout on community analysis of phenotype data), and perform community analysis of their whole microbiome sequencing data (see handouts corresponding to community analysis of sequencing data for week 6 of the half-semester implementation). The half-semester implementation allows insufficient time for students to learn to use DNA Subway to create a taxonomy table for subsequent community ecology analysis. Therefore, it is necessary for the instructor or teaching assistant to perform the data processing in DNA Subway and present students with the resulting taxonomy table for analysis. As in the full-semester implementations, students can present their results in the final week of the project in either the written or oral formats suggested above.
While still in its early stages, microbiome research has grown rapidly and garnered the attention of both scientists and non-scientists alike, providing an excellent and engaging topic area for undergraduate research experiences. Inquiry-based activities for student microbiome research have previously been developed for aquatic ecosystems (
The Bean Beetle Microbiome CURE is aimed to provide students with an authentic research experience as part of the course curriculum that enables them to develop skills related to the scientific process. Studies have shown that research experiences provide students with a greater sense of autonomy and ownership of their projects (
An important component of our current research on the BBMP-CURE is the level of student autonomy, or the level of responsibility, related to the development of the research question addressed. Our approach provides two levels of autonomy that we categorize as either low-autonomy (faculty-driven questions) or high-autonomy (student-driven questions). While we hypothesize that a greater level of student autonomy will have a positive effect on student outcomes, it is anticipated that students will strengthen their science-process skills regardless of the level of autonomy chosen, as they analyze the results of their experiments, draw conclusions, connect their research to the broader literature, and communicate their results. Our ongoing research based on assessments of survey data for past and future implementations of the BBMP-CURE will be used to test this hypothesis.
In the fall and spring semesters of the 2019–2020 academic year, a total of 10 faculty participants from six different institutions (most of which were minority-serving institutions) implemented the BBMP-CURE (
Despite the reported educational benefits of CUREs, challenges to their implementation persist (
Another challenge of CUREs is that the authentic nature of the research means that, like with all research, students likely will carry out procedures that do not work in the first attempt. Research is inherently an iterative process where scientists must navigate scientific challenges, persevere through difficulties, and cope with failure (
The most significant barrier to incorporating microbiome research for undergraduate education is the cost associated with next-generation sequencing (NGS) (
Other challenges may arise in the sessions dedicated to sequence data analysis using computational methods. Depending on the classroom set up, certain technical difficulties may arise (e.g., slow/unreliable internet connections, students who do not own personal laptops, students with different levels of familiarity with computers or spreadsheets). A teaching assistant or peer-mentor who can move around freely to aid students who may be struggling can be a valuable asset for these laboratory sessions. Additionally, access to university computational resources (e.g., computer labs) may alleviate some of the challenges of performing computational analysis in the laboratory classroom. For the DNA Subway portion of the bioinformatic analysis, students are encouraged to work in groups such that only one computer per group is logged on to the server to perform the metagenomic analysis, thereby reducing demands on the server that might prolong time to completion of the analysis. Alternatively, faculty may opt to have students perform the analyses as outside classwork so that students can have ample time to complete the analyses. Written and video tutorials are available to facilitate students’ independent remote learning
Finally, while the BBMP-CURE was originally designed for hands-on laboratory instruction, the global outbreak of COVID-19 has significantly altered current instructional programs. It is uncertain when and to what extent opportunities for in-person instruction will become available again for all undergraduates. The current situation facing universities underscores the need for quality classroom activities that can be performed in online and virtual formats. The work presented here adds value to the current state of instruction by providing easy-to-implement modules with access to our online-database of isolate and whole-community sequence datasets, which allows for virtual implementation of many aspects of this CURE.
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/
The studies involving human participants were reviewed and approved by Emory University Institutional Review Board (IRB00113934). The patients/participants provided their written informed consent to participate in this study.
AZ performed literature review, created handouts and instructional materials, and wrote and edited majority of the manuscript. NG, LB, and CB created handouts and instructional materials and wrote and edited the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors would like to acknowledge the participants of the 2019–2020 Bean Beetle Microbiome Project for providing invaluable feedback on the implementation of this CURE.
The Supplementary Material for this article can be found online at:
Survey data collected from faculty participants of the Bean Beetle Microbiome Project for the 2019–2020 academic year.
Course timeline for full-semester implementation.
Course timeline for half-semester implementation.
Example student and/or faculty-generated research questions implemented in the 2019–2020 academic year.
Preliminary results of group differences on the Laboratory Course Activities Survey (LCAS).
Mean and standard deviation for student responses from the Persistence in the Sciences (PITS) Survey.