A research program-linked, course-based undergraduate research experience that allows undergraduates to participate in current research on mycobacterial gene regulation

Abstract

Undergraduate instructional biology laboratories are typically taught within two paradigms. Some labs focus on protocols and techniques delivered in "cookbook" format with defined experimental outcomes. There is increasing momentum to alternatively employ student-driven, open-ended, and discovery-based strategies, often via course-based undergraduate research experiences (CUREs) using crowd-sourcing initiatives. A fraction of students also participate in funded research in faculty research labs, where they have opportunities to work on projects designed to expand the frontiers of human knowledge. These experiences are widely recognized as valuable but are not scalable, as most institutions have many more undergraduates than research lab positions. We sought to address this gap through our department's curriculum by creating an opportunity for students to participate in the real-world research process within a laboratory course. We conceived, developed, and delivered an authentic, guided research experience to students in an upper-level molecular biology laboratory course. We refer to this model as a "research program-linked CURE." The research questions come directly from a faculty member's research lab and evolve along with that research program. Students study post-transcriptional regulation in mycobacteria. We use current molecular biology methodologies to test hypotheses like "UTRs affect RNA and protein expression levels," "there is functional redundancy among RNA helicases," and "carbon starvation alters mRNA 5' end chemistries." We conducted standard assessments and developed a customized "Skills and Concepts Inventory" survey to gauge how well the course met our student learning outcomes. We report the results of our assessments and describe challenges addressed during development and execution of the course, including organizing activities to fit within an instructional lab, balancing breadth with depth, and maintaining authenticity while giving students the experience of obtaining interpretable and novel results. Our data suggest student learning was enhanced through this truly authentic research approach. Further, students were able to perceive they were participants and contributors within an active research paradigm. Students reported increases in their self-identification as scientists, and a positive impact on their career trajectories. An additional benefit was reciprocation back to the funded research laboratory, by funneling course alumni, results, materials, and protocols.

Keywords: CRISPRi; Mycolicibacterium smegmatis; RNA; assessments; authentic research; gene expression; molecular biology; undergraduate laboratory teaching.

Figure 1

Workflow for creating an rpl-CURE.

Figure 2

General timeline of student experiments and course assessments.

Figure 3

SCI learning gains, comapring in-person (n = 16 student responses) vs. entirely remote (n = 17) delivery for the MBGE lab.

Figure 4

Research thrust 1: Using reporters to evaluate 5′ UTR roles. (A) Plasmids used to assess the effects of 5′ UTRs on expression of the reporter gene yfp. The P_myc1-tetO promoter was used in the absence of a tet repressor to achieve constitutive high-level transcription. Plasmid elements not shown include and E. coli origin of replication, an antibiotic resistsnce marker, and sequences to integrate the plasmid into the Mycolicibacterium smegmatis chromosome by site-specific recombination. A plasmid with the P_myc1-tetO associated 5′ UTR (left, pSS303) served as a positive control for efficient translation. Students used HiFi assembly (NEB) to insert M. smegmatis 5′ UTRs of intrest into a plasmid lacking a 5′ UTR (right, pSS310). Graphics created with BioRender.com. (B) Student workflow for plasmid construction, strain construction and expression testing. An example of data generated by students is shown.

Figure 5

Research thrust 2: Using CRISPRi to access functional redundancy in RNA degradation proteins. (A) CRISPRi plasmids used to express catalytically dead Cas9 and sgRNAs. Students used HiFi assembly (NEB) to replace part of a non-targetting sgRNA with gene-specific sequence. Plasmid elements not shown include dcas9, an E. coli origin of replication, and anibiotic resistence marker, and sequence to integrate the plasmid into the M. smegmatis chromosome by site-specific recombination. Expression of the sgRNA and dCase9 was controlled by ATc-inducible promoters. Graphics created with BioRender.com. (B) Student workflow for plasmid construction, strain construction, and measuring growth. An example is shown of student data testing for functional redundancy between the RNA helicases helicases HeIY and RhIE1.

Figure 6

Research thrust 3: Assessing mRNA 5′ end cap state by splinted ligation. (A) The relative proportion of monophosphorylated mRNA 5′ ends was assessed by measuring the efficiency of ligation to an adaptor, which is dependent upon the presence of a 5′ monophosphate. mRNAs with 5′ triphosphates or other 5′ end chemistries are not ligatable. Primers that anneal internally within the transcript were used to quantify total transcript, and an adapter primer paired with and internal primer were used to quantify ligated transcript. (B) Student workflow for confirming the deletion of putative mRNA 5′-end acting genes in M. smegmatis strains obtained from a large collection (Judd et al., 2021), designing splints and primers to assess the 5′ end chemistry of putative target transcripts, optimizing standard and carbon starvation culture conditions, extracting RNA, and performing splinted ligation and PCR-based analysis of the ligation products. An example is shown of student data testing the impact of a gene deletion and carbon starvation on the ligatability of a target transcript.