Improving Student Outcomes with an Adaptable Molecular Cloning Course-Based Undergraduate Research Experience

Summary

An adaptable Gibson Assembly molecular cloning module was employed in a course-based undergraduate research experience (CURE) format for molecular biology laboratory course students. Assessment of student learning outcomes showed improved understanding and confidence in molecular cloning after completion of the CURE, and novel plasmids were cloned for natural product biosynthesis research.  

Abstract

The continuous advancement of molecular biology techniques requires that molecular biology curricula are regularly refined to effectively prepare students to enter the workforce with modern competencies. In particular, the emergence of Gibson Assembly, a highly customizable and adaptive molecular cloning technique, has advanced the landscape of molecular cloning in numerous research environments. Thus, we created a Gibson Assembly cloning module for deployment in a molecular biology laboratory course at California Polytechnic State University, San Luis Obispo and evaluated student learning outcomes from the module. Over three iterations of the course, students participated in an experiment-based independent project that involved cloning three unique plasmid libraries to support research projects in natural products biosynthesis. Students were given pre- and post-questionnaires to evaluate their understanding of molecular cloning and their confidence in molecular biology terms and techniques. Students’ responses showed a significant increase in both learning molecular cloning concepts and in self-reported confidence with molecular cloning terms and techniques. This module framework can be generalized to teach Gibson Assembly for various applications, providing instructors with a toolkit for teaching an adaptable and emergent cloning technology while advancing their research projects.

Introduction

Training undergraduates in fundamental molecular biology concepts and laboratory techniques is crucial for their scientific and professional development as these methodologies are common in various research settings, including academia and industry. As such, students in the biology (molecular and cellular biology concentration) and biochemistry majors at California Polytechnic State University, San Luis Obispo (Cal Poly) are required to take an upper-division molecular biology laboratory course to learn and apply these topics (CHEM/BIO 475). A base curriculum for this course has been previously developed wherein students perform topoisomerase-based (TOPO) cloning to a....

Protocol

The studies involving human participants were reviewed and approved by human subjects in the Research Institutional Review Board at Cal Poly (2022-113-CP (IRB)). The participants provided their written informed consent to participate in this study.

The following protocol outlines instructor preparation (steps 1.1–1.3), students’ actions for a three-experiment teaching module that includes PCR to obtain linear fragments (steps 2.1–

Discussion

Here, we propose an adjustable undergraduate classroom laboratory project that teaches students molecular cloning through Gibson Assembly in a course-based research setting. In total, 28 novel plasmids were cloned by undergraduate students in a classroom setting. Individual student-reported success ranged from 44.4% to 81.8% over three cohorts, and overall cloning success was 80% (28 of 35 total assigned plasmids were successfully cloned). Lower cloning success in 2023 was likely due to two compounding factors. First, tu.......

Materials

Name	Company	Catalog Number	Comments
Deoxyribonucleotide triphosphate (dNTPs, 10  mM)	Fisher Scientific	FERR0191	Homemade' MasterMix component
Dithiothreitol (DTT)	Fisher Scientific	FERR0861	Homemade' MasterMix component
DpnI	New England Biolabs	R0176S	1000 units
Fisherbrand Isotemp Microbiological Incubator	Fisher Scientific	15-103-0513
FisherBrand Isotemp Water Bath	Fisher Scientific	S28124
GelRed Nucleic Acid Gel Stain	Biotium	NC9594719	10,000X
GeneJET Gel Extraction and DNA Cleanup Micro Kit	Thermo Scientific	FERK0831	100 Preps
GeneRuler 1 kb DNA ladder	Fisher Scientific	FERSM0314	100 applications
LB Broth, Miller	Fisher BioReagents	BP9723-500	500 g
Magnesium chloride hexahydrate	Fisher Scientific	BP214-500	Homemade' MasterMix component
Mastercycler nexus X2 Gradient Thermocycler	Eppendorf	6337000027
Microfuge 16 Centrifuge	Beckman Coulter	A46474
Micromonospora echinospora bacteria	American Type Culture Collection	ATCC 15837
Microwave Oven	General Electric	2440640
Molecular Biology Grade Agarose	Fisher BioReagents	BP160-100	100 g
Nanodrop One Microvolume Spectrophotometer	Thermo Scientific	13-400-518
NEB 5-alpha Competent E. coli	New England Biolabs	C2987H	20 x 0.05 mL
NEBuilder HiFi DNA Assembly Master Mix	New England Biolabs	E2621S	10 reactions
New Brunswick Innova 40 Benchtop Orbital Shaker	New Brunswick	M1299-0090
Nuclease Free Water	Fisher BioReagents	BP248450	50 mL
PEG-8000	Fisher Scientific	BP233-100	Homemade' MasterMix component
Phusion DNA Polymerase	New England Biolabs	M0530	Homemade' MasterMix component
Portable Balance	Ohaus	SKX123
pUC19 vector	New England Biolabs	N3041S
Q5 High-Fidelity 2x Master Mix	New England Biolabs	M0492S	100 reactions
T5 Exonuclease	Epicentre	T5E4111K	Homemade' MasterMix component
Taq DNA Ligase	New England Biolabs	M0208	Homemade' MasterMix component
Tris-HCl	Fisher Scientific	AAA1137918	Homemade' MasterMix component
TriTrack DNA Gel Loading Dye (6x)	Thermo Scientific	FERR1161	5 x 1 mL
Zyppy Plasmid Miniprep Kit	Zymo Research	D4019	100 Preps
β-Nicotinamide adenine dinucleotide (NAD+)	New England Biolabs	B9007S	Homemade' MasterMix component