Development of a Remote, Course-Based Undergraduate Experience to Facilitate In Silico Study of Microbial Metabolic Pathways.

Course-based undergraduate research experiences (CUREs) often occur in a physical lab space, but they can also be offered remotely while maintaining course expectations and providing opportunity for authentic student engagement in research. Using a novel framework, remote Microbial Ecology CURE students used microbes isolated via antimicrobial-challenged Winogradsky columns to investigate phylogeny and metabolism through a hypothesis-driven meta-analysis (MA). Students used 16S rRNA and key metabolic enzymes to compare phylogeny; enzymes were modeled and evaluated for putative conserved domains, culminating in primer design and analysis. Using in silico tools facilitated student development of bioinformatics skills. The MA was subdivided into discrete sections in order to (i) provide a timeline for students to remain on schedule throughout a remote-learning lab experience, (ii) encourage feedback throughout the project, and (iii) facilitate student understanding of the experimental design. MA deliverables were designed to be specific figures with individual titles, legends, and analyses to enable their feedback for subsequent presentations. The six key formative deliverables included a word cloud (used to develop the works cited list and hypothesis), a 16S rRNA phylogenetic tree, an annotated metabolic pathway and three-dimensional model of the key metabolic enzyme, a phylogenetic tree based on the key metabolic enzyme, design and analysis of a primer set for the key metabolic enzyme, and a summative poster and graphical abstract. The MA project yielded poster presentations at virtual conferences, lab presentations, and written reports. Using the hypothesis-based MA model encouraged an authentic research experience, enabling students to develop, discuss, and progress in meaningful experiments.

INTRODUCTION

There is consistent need for authentic research experiences for undergraduates (1), including a significant need for bioinformatics, modeling, and familiarization with such analyses (2–5). Novel course-based undergraduate research experiences (CUREs) provide such opportunities (6, 7).

The Microbial Ecology CURE (ME-CURE) was designed for upper-level undergraduate students to investigate antimicrobial effects of essential oils, herbs, and spices upon soil microbes. Previously, face-to-face (F2F) ME-CURE students selected compounds with reported antimicrobial activity (i.e., turmeric), added such compounds to Winogradsky columns (WC) (8), and isolated microbes from the columns using media supplemented with the compound, yielding >100 isolates. Previous F2F ME-CURE students characterized such isolates phylogenetically (Gram staining and 16S rRNA sequencing) and analyzed isolate antimicrobial susceptibility to both the compound and commercially available antibiotics (disc diffusion and Kirby-Bauer assays) (8). Prior F2F analyses prompted further evaluation of microbial mechanisms, including metabolic pathways, enabling growth on such compounds. In silico tools evaluating antimicrobial compounds, including bioavailability, 50% lethal dose (LD50), and toxicity, have been reported (9). While there is guidance for inclusion of bioinformatics in undergraduate education (10), there remain significant concerns in the ability to do so (11).

Previous ME-CURE students isolated and characterized bacteria from WC challenged with turmeric (12), contributing to questions related to metabolism of, or avoidance of the antimicrobial effects from, turmeric. Such work led to student investigation (Appendix 1) related to turmeric and the associated curcumin degradation pathway, including primer design targeting curA (a gene related to curcumin metabolism in Escherichia coli) (13) in such isolates (12, 14, 15). Based on the successful in silico analysis of the turmeric pathway and curA, a meta-analysis (MA) project was designed for implementation in a remote ME-CURE.

PROCEDURE

For the online CURE, students were instructed to select an antimicrobial compound (from those used in prior ME-CUREs) in order to explore potential pathways used by isolates to grow in the presence of such compounds. In order to facilitate student success in the remote CURE, a MA project was developed to guide students through a meaningful and rigorous dry lab experience. The experimental and learning goals were to (i) identify a potential metabolic pathway related to the compound, (ii) investigate a potential key enzyme from the pathway, (iii) analyze a three-dimensional (3D) model of the key enzyme to identify conserved residues, and (iv) develop primers for future identification of similar enzymes in lab isolates. Incorporation of bioinformatics was through 16S rRNA and amino acid alignments, development of phylogenetic trees, and subsequent analysis. The experimental flow (Fig. 1) guided students through the MA while providing clear deadlines and assessment parameters. Learning goals were accomplished through a 6-part MA. Students were placed into Slack channels to facilitate group work and centralized lab channels (e.g., websites_tools and journal_club) for discussion and troubleshooting.

FIG 1

Experimental flow for the Microbial Ecology CURE meta-analysis. Clear goals for students in the lab were established prior to the start of semester such that the lab schedule was organized to best facilitate student success.

Breaking the MA into discrete deliverables provided clear objectives to facilitate the CURE. Students were given detailed rubrics and guidelines for the entire project at the start of the semester (Appendix 2a to g) and provided with MA examples throughout the semester (Appendix 1). In silico tools provided to students were deliverable-specific (Table 1) and facilitated student development of lab methods. Lab groups conducted literature reviews related to previously used compounds from the lab. The first deliverable (MA 1) included development of a key terms word cloud, a central hypothesis for the MA, and identification of a metabolic pathway related to the compound of interest. Students used 16S rRNA sequences from F2F ME-CURE semesters, coupled with published sequences, to develop phylogenetic trees of microorganisms reportedly susceptible or resistant to the compound (MA 2). Students conducted a 3D analysis of putative key enzymes from the metabolic pathways (MA 3) and amino acid BLAST analysis, alignment analysis, and phylogenetic analysis of the key enzyme (MA 4). Such analyses facilitated primer design for the key enzyme (MA 5). The final deliverable was a summative graphical abstract and poster presentation (MA 6) (Fig. 1).

TABLE 1

Discrete deliverables and suggested in silico programs and websites

Select meta-analysis deliverable(s)	Delivery timeline (wk due per 15-wk semester)	Suggested programs and/or websites
Word cloud	Wk 3	https://www.wordclouds.com/, https://monkeylearn.com/word-cloud/, https://www.freewordcloudgenerator.com/generatewordcloud, https://coolinfographics.com/word-clouds
Labeled and annotated metabolic pathway	Wk 3	https://biocyc.org/META/organism-summary, https://www.genome.jp/kegg/pathway.html
16S rRNA phylogenetic tree	Wk 6	https://www.phylogeny.fr/, http://rdp.cme.msu.edu/, https://www.megasoftware.net/
Protein structure analysis	Wk 7	https://www.ncbi.nlm.nih.gov/Structure/MMDB/mmdb.shtml, https://www.ncbi.nlm.nih.gov/Structure/lexington/lexington.cgi, http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index
Protein phylogenetic tree	Wk 8	https://www.phylogeny.fr/, https://www.megasoftware.net/
Primer set and analysis	Wk 13	https://www.ncbi.nlm.nih.gov/tools/primer-blast/, https://www.idtdna.com/pages/tools/oligoanalyzer, https://www.idtdna.com/pages/tools/primerquest, https://bioinfo.ut.ee/primer3-0.4.0/
Graphical abstract	Wk 14	https://www.elsevier.com/authors/tools-and-resources/graphical-abstract, https://www.annaclemens.com/blog/make-graphical-abstract-paper, https://www.cell.com/pb/assets/raw/shared/figureguidelines/GA_guide.pdf, https://medium.com/the-science-educator/writing-a-scientific-paper-check-our-graphical-abstract-templates-7b6ab3cb4720, http://users.eecs.northwestern.edu/~jhullman/graphical_abstracts.pdf, https://docs.google.com/document/d/1yxEUwzBLJfnpZB9Y85CXnP_zCkj6loKdZyZHyMloRyY/edit

Constructive feedback from the instructor, teaching assistant, and ME-CURE peers provided students with the ability to review, modify, and improve their writing and analyses throughout the semester. Students were required to present their poster twice, at a minimum, in the lab and through the Georgia State University (GSU) STEM conference. Students were encouraged to revise their posters for additional conferences, including the GSU Undergraduate Research Conference (GSURC) and Southeastern Branch American Society of Microbiology (SEBASM).

CONCLUSION

The remote ME-CURE built upon prior F2F ME-CURE findings. While work progressed in previous lab semesters, isolates from varied antimicrobial-supplemented WC only began to tell the story of such microbes, microbial growth, and the potential for antimicrobial resistance/sensitivity. After several semesters of bacterial isolation, it became necessary to consider how best to approach further investigation of the microbes, as well as characterization of how the antimicrobial compounds were rendered ineffective in the isolates. This realization was concurrent with the shift to online labs in Spring 2020.

The development of the MA project was based upon several academic, research, and professionalism needs, as well as the need to incorporate bioinformatics into the remote ME-CURE. There were needs for the (i) development of online CUREs to support remote research opportunities for GSU students (1), (ii) incorporation of bioinformatics tools to facilitate the learning of microbial metabolic pathways and design of pertinent primers to identify microbial isolates from previous CUREs capable of metabolism and/or degradation of compounds of interest, and (iii) development of an assignment that would promote student professionalism through timed deliverables and intermittent, semester-long feedback.

Establishment of clear deliverables and rubrics facilitated a pipeline through which students could advance their research, become comfortable with bioinformatics tools, and develop STEM literacy and presentation skills. Intellectual merit gained through the ME-CURE collaborative effort is significant and represents novel contributions to the ME field. At the end of the semester, one student commented, “I like how we do everything in, like, little chunks, and then it all comes together…A [GSURC] judge that came and looked at our research said, ‘Wow! You guys have done a lot of research,’ but it didn’t feel that way because it was all broken down and then it all came together.” Such activity helped to encourage students to share ideas and become more proficient with hypothesis development, analysis, and presentation of their results. This proficiency was exemplified by the majority of students presenting several times throughout the semester, with 100% participation in the GSU STEM Conference, 75% participation in GSURC, and one presentation at the SEBASM Conference (15). The use of this scaffolded MA can be modified to facilitate both F2F and remote learning of phylogenetic analysis and metabolic pathways through in silico tools. Facilitation of student analysis and experimentation, using compounded deliverables, enabled successful presentations, developed group dynamics, and furthered STEM literacy, while incorporating bioinformatics into the ME-CURE.