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

Christopher B. Cummings; Samuel S. Catania; Eirene M. Q. Ednacot; Austin J. Kinsella-Johnson; Claire E. Meeds; Jack W. Reynolds; Ava E. Sanderson; Rachel A. Johnson; Katharine R. Watts

doi:10.3791/67067

JoVE Journal > Biochemistry

Biochimie

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

Published: November 15, 2024

doi:

10.3791/67067

Christopher B. Cummings*^1,2, Samuel S. Catania*^1,2, Eirene M. Q. Ednacot^1,2, Austin J. Kinsella-Johnson^1,2, Claire E. Meeds^1,2, Jack W. Reynolds^1,2, Ava E. Sanderson^1,2, Rachel A. Johnson^1,2, Katharine R. Watts^1,2

¹Department of Chemistry and Biochemistry,California Polytechnic State University, ²Center for Applications in Biotechnology,California Polytechnic State University

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 assemble an actin-containing plasmid that is prepared from a yeast complementary DNA (cDNA) template¹. Students design experiments based on questions that mimic authentic research hypotheses, increasing their familiarity with laboratory practices and inquiry-based learning. Continuous advancement in the field of molecular biology requires that corresponding curricula adapt to prepare students with modern competencies for the workforce. In particular, the use of Gibson Assembly has become more predominant in the scientific community; while the method was originally established to synthesize artificial chromosomes², over 5,000 publications at the time of this report have referenced Gibson et al.’s original work. Gibson Assembly is unique compared to traditional cloning methodologies: it is highly customizable and can easily ligate multiple linear DNA fragments without the need for restriction sites to produce the junctions. Thus, we saw the opportunity to revamp the CHEM/BIO 475 curriculum to incorporate modern molecular cloning techniques and improve the inquiry-based course model.

It has been established that student research experiences contribute to increased conceptual understanding, skill development, and persistence in science³, yet not all undergraduate students have the opportunity to participate directly in a research laboratory. To address the challenge of limited student capacity in research laboratories, course-based undergraduate research experiences (CUREs) have been developed and employed to increase science accessibility through authentic research in the classroom. While CUREs vary in their implementation, common practices that address the five tenets of scientific research have been established. In a well-designed CURE, students will 1) use scientific practices, 2) collaborate in a research project, 3) attempt to make new discoveries, 4) contribute to work relevant outside of the classroom, and 5) reassess and revise hypotheses and methods in the case of experimental failure⁴. Similar to traditional student research experiences in a laboratory, CUREs have been shown to strengthen student confidence in science, scientific skills, project ownership, and persistence in science, technology, engineering, and mathematics (STEM)⁵. While CUREs involving molecular cloning have been reported previously⁶^,⁷^,⁸^,⁹^,¹⁰^,¹¹^,¹²^,¹³, we are unaware of any that emphasize the adaptability of Gibson Assembly to make a library of authentic research plasmids.

Here, we report an expansion of the current inquiry-based CHEM/BIO 475 curriculum at California Polytechnic State University, San Luis Obispo with two major improvements: hands-on experience using Gibson Assembly and student participation in a CURE, which has provided original plasmid constructs for research projects funded by the National Science Foundation (NSF-1708919 and NSF-2300890). Over three implementations of this curriculum, students have contributed to two distinct research projects focused on natural product biosynthesis of bioactive molecules produced by Actinomycetota. Natural products often contain pharmacophores with antibiotic, antifungal, and/or anticancer activities, giving these small molecules importance in drug discovery efforts and potential for clinical relevance¹⁴. This research requires the creation of plasmid libraries to enable investigations of both the function and engineering potential of bacterial biosynthetic enzymes. In this CURE, students designed and performed Gibson Assembly experiments to clone the unique plasmid libraries pertinent to these research projects (Figure 1). In addition, the format and design of the module is distinctive because it is easily adaptable to generate any plasmids of interest for other research projects.

Figure 1: Overview of the role of Gibson Assembly in our research laboratory. Actinomycetota produce small molecule natural products with clinically relevant bioactivities using gene clusters that encode biosynthetic enzymes. In our research, plasmids containing a biosynthetic gene are assembled via Gibson Assembly for downstream investigations of the encoded enzyme’s function. Science icons from Biorender.com. Please click here to view a larger version of this figure.

Laboratory overview

Molecular Biology Laboratory is an upper-division course required for biochemistry majors and biology majors with a molecular and cellular biology concentration. Other students meeting prerequisites are welcome to take the course as an upper-division elective. The course is co-listed between the Chemistry and Biochemistry Department and the Biology Department at Cal Poly. Faculty from both departments take turns teaching the course each term (two quarters Biology, one quarter Chemistry and Biochemistry).

The class meets in the laboratory twice each week for 170 min periods and once a week for a 50 min lecture. Lab sections contain a maximum of 16 students and 2–3 lab sections are offered each quarter. The course runs for 10 weeks, and the final exam is administered during the last meeting in week 10. Lecture time is spent discussing the theory behind many of the experimental techniques conducted in the lab, as well as current topics in molecular biology that are not covered in the lab. The core curriculum of the lab encompasses the process of cloning the actin gene from yeast¹, which takes approximately 7 weeks (13–14 lab meetings). Techniques include micropipetting, yeast RNA isolation, amplifying a yeast gene using reverse transcription-polymerase chain reaction (RT-PCR), TOPO cloning, blue-white screening, plasmid isolation and verification of insert by restriction digest and PCR, in silico analysis of clones, and DNA sequence analysis. The curriculum for the last 3 weeks of the course is at the discretion of the instructor but involves students completing an “independent project” with undefined outcomes.

Experiment overview

One focus of our research group is biosynthetic pathways in Actinomycetota. When designing the independent project, we envisioned having students create plasmids through Gibson Assembly for use in our research projects investigating natural product biosynthesis. While the iterations of the course module that are assessed here were particular to plasmids that allow for the manipulation of biosynthetic pathways, the Gibson Assembly workflow is enormously adaptable for other molecular cloning projects (Figure 2). The workflow was split into three different experiments (A, B, and C) that were completed over two lecture periods and six lab periods (3 weeks total) (See Supplemental File 1 and Supplemental File 2). Experiments were preceded by worksheets to support student preparation and assess student understanding (Supplemental File 3, Supplemental File 4, and Supplemental File 5). The workflow is presented in a format that is flexible to an instructor’s needs and interests.

Figure 2: Gibson Assembly module workflow. Day 1 and Day 4 are lecture periods where students complete in silico sequence analysis and experimental design. Days 2–3 and 5–8 are lab meetings where the steps to clone novel plasmids via Gibson Assembly are conducted, followed by isolation and screening. The pictorial flowchart is grouped by the three experiments students perform (A, B, and C). More detailed directions and protocols can be found in the Instructor and Student Manuals provided as Supplemental File 1 and Supplemental File 2, respectively. Science icons from Biorender.com. Please click here to view a larger version of this figure.

The Gibson Assembly independent project module was first piloted in the spring quarter of 2019 in CHEM/BIO 475. In 2020 and 2021, the course was taught online due to the SARS-CoV-2 pandemic. When in-person instruction resumed in the Spring of 2022 and 2023, students within the course were invited to participate in a study assessing learning outcomes of a Gibson Assembly independent project where original, research-relevant plasmids would be cloned. In 2019, students created a library of plasmids consisting of gene cassettes from the genome of Micromonospora echinospora ATCC 15837 that were cloned into pKC1132 (Figure 3). This plasmid library is being utilized in our research lab to inactivate genes of interest in a putative biosynthetic gene cluster for the natural product TLN-05220¹⁵. To complement our gene inactivation studies, students in 2022 cloned a small library of genes from the putative TLN-05220 gene cluster into pUC19 (Figure 3); our research group has utilized these plasmids for subcloning genes into expression vectors, including pET28b, for protein overexpression and purification. Students in the 2023 cohort contributed to ongoing work on a biosynthetic engineering project on the epoxomicin synthetase¹⁶. In teams of 3–4, students cloned engineered domains of non-ribosomal peptide synthetase¹⁷ modules into various protein expression vectors to optimize the overexpression and purification of these enzymes in our research lab (Figure 3). Redundancy was built into the cloning plan for each cohort. For example, the 2019 cohort contained 44 students, and 15 plasmids were assigned to the class, for cloning. Thus, cloning of each plasmid was attempted two or three times.

Figure 3: Summary of plasmids cloned and student participants during 2019, 2022, and 2023 independent project iterations. The Gibson Assembly project has been deployed three times. In each offering, student participants cloned a different library of plasmids to be utilized in research projects exploring biosynthetic pathways. Projects in 2019 and 2022 supported our ongoing work on the natural product TLN-05220¹⁵ with two fragment (one gene cassette or gene and a vector) Gibson Assembly reactions. The 2023 project involved domain swapping within modules 1 and 2 of a non-ribosomal peptide synthetase (NRPS) enzyme that is involved in epoxomicin biosynthesis¹⁶. The hatched fragments represent two different mutants of the swapped domain, and solid colors represent domains that were not swapped. In total, eight different gene fragments (four for module 1 and four for module 2) were generated with overhangs compatible for Gibson Assembly. For each module, two different combinations of three gene fragments were assembled with one of two different vectors (pBAD33 and pET28, four total fragments per assembly), for the potential to generate eight engineered NRPS plasmids. Science icons from Biorender.com. Please click here to view a larger version of this figure.

Assessment overview

At least 1 week prior to the start of the Gibson Assembly independent project, students in the 2022 and 2023 cohorts were invited to participate in a learning outcome investigation, designed similarly to a study performed in a survey of biochemistry lab course at Cal Poly¹⁸. Student participants completed a multiple-choice pre-questionnaire in the lab meeting before the start of the independent project and a multiple-choice post-questionnaire during the final lab meeting (i.e., after they had completed the Gibson Assembly module). The pre- and post- questionnaires consisted of 28 identical questions with two additional questions in the post-questionnaire (30 total). Ten content questions were written to assess students’ knowledge of enzymes and mechanisms involved in molecular cloning (e.g., polymerase chain reaction [PCR], Gibson Assembly, transformation, blue-white screening). The following seven questions asked students to self-assess their familiarity with molecular cloning terms (e.g., DNA polymerase, exonuclease, ligase). The next 10 questions allowed students to self-assess their ability to perform molecular cloning techniques (e.g., DNA sequence analysis, restriction digest reactions, agarose gel electrophoresis). Students also reported whether they would be comfortable pursuing a career in molecular biology based on their knowledge of molecular cloning techniques. Two additional questions were included in the post-questionnaire for students to self-assess their commitment to learning in the course and whether the course was a valuable learning experience (Supplemental File 6 and Supplemental File 7). All data from the 2022 and 2023 student pre- and post-questionnaire responses were combined for analysis and are available in Supplemental Table S1.

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 2.1–2.7), Gibson assembly, transformation, and selection of clones (steps 3.1–3.5), plasmid isolation and screening (steps 4.1–4.4), and assessment of learning outcomes (5.1–5.2). Instructor preparation describes one representative example of primer design and preparation of a desired plasmid map in silico. All sections of the protocol are adaptable for other desired plasmids. Each student experiment is divided into two 3 h lab meetings.

1. Instructor preparation

Gibson Assembly primer design using Benchling Assembly Wizard
1. Determine the DNA template source for the gene insert(s), such as genomic DNA, plasmid and synthetic DNA.
  NOTE: For genes that will be amplified from complex genomic DNA templates, it is advised to first determine unique binding sites for primers within the genomic DNA template that are specific for the gene(s) of interest, using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST)²¹, as detailed in Supplemental File 1. For simple templates such as plasmids or synthetic DNA, proceed to the next step.
2. Retrieve DNA sequences of the gene insert(s) and vector (for example, see polyketide hydroxylase gene sequence and pUC19 sequence provided as GenBank files in the Supplemental File 8). Import the desired insert sequence(s) and desired vector sequence to Benchling, each as a new DNA sequence file. Open each imported sequence to be included in the Gibson Assembly reaction.
  NOTE: One can import DNA sequence files in several formats, including GenBank and FASTA. One can also import a sequence directly from a database with an accession number or paste in a nucleotide sequence copied from another file or viewer.
3. At the bottom of the screen, locate the Assembly Wizard tool. Click Assembly Wizard, then select Create New Assembly. From the options provided, select Gibson and click Start to begin the assembly.
4. In the vector sequence window, select all bases to be included in the assembly from the vector backbone. Start the selection at the nucleotide position at the 3’ end of the gene insert (e.g., position 657 of pUC19), and select all remaining nucleotides to be included (e.g., through position 656 to include entire pUC19 sequence). Once selected, click the Backbone tab at the bottom of the screen| Set Fragment.
  NOTE: Toggle between the Sequence Map and Plasmid views of the vector to select all desired bases of a circular template. Alternatively, if planning to prepare the vector fragment via restriction digest (instead of PCR) for Gibson Assembly, linearize the backbone sequence with a restriction enzyme cut site (e.g., XbaI). To do so, click the cut site, hold Shift, and click the cut site again. Then click Set Fragment to set the backbone. Be aware that vector-specific primers will not be generated by Benchling if the backbone is linearized with a restriction enzyme in the Assembly Wizard.
5. In the insert sequence window, select all the insert's bases to be included in the assembly. Once selected, click the Insert tab at the bottom of the screen | Set Fragment.
6. In case of multiple gene inserts, click the + button on the right side of the Assembly Wizard. In the insert sequence window, select all the insert bases to be included in the assembly. Once selected, click the Insert tab at the bottom of the screen | Set Fragment.
7. Once all fragments are set, rename the assembly to the desired plasmid name and click Assemble on the right of the Assembly Wizard. Select the desired folder location for both the Sequence Folder and Primer Folder. Click Create to assemble the recombinant plasmid sequence.
8. Open the assembled plasmid and click Assembly History to view a basic plasmid map and an overview of the sequences from which it was derived. Ensure that the nucleotide positions for each fragment match the desired design.
9. In the Assembly Parameters tab, view the names of the primers designed for the assembly. Note that there will be two primers designed for each insert (a forward and a reverse), and the primer names will be derived from the titles of the DNA sequence files. Also ensure that primer melting temperatures and suggested annealing temperatures are compatible in this window (see Supplemental File 8, Representative Primers).
  1. If primer melting temperatures or annealing temperatures are not ideal, manually adjust primer sequences (e.g., removing nucleotides to reduce melting temperature) within the primer sequence files. Alternatively, click on Re-open and adjust the primer settings by clicking the Enzyme/Primer Settings button next to the Assemble button in the Assembly Wizard. Then, repeat the Assembly step and re-examine primer sequences.
  2. If a library of plasmids is to be cloned with many different genes cloned into the same vector backbone,
  3. Utilize the same primer pair for the vector in each case. To do so, manually remove all the nucleotides from the vector primers that would install the overhangs that are specific to the insert. These nucleotides would be found at the 5’ end of the primer. After removal, ensure that primer melting temperatures and annealing temperatures are compatible (see Supplemental File 8, Representative Primers).
  4. Ensure all insert primer pairs have very similar melting temperatures and annealing temperatures.
    NOTE: This will reduce the need for annealing temperature optimization of every pair and allows for all student PCR reactions to be cycled at the same time using the same thermocycler program.
10. Once primer design is complete for desired plasmids, click Finalize in the Assembly Wizard. Order the primers from a DNA synthesis company and retrieve samples of the DNA templates for insert(s) and vector.
11. Test both insert- and vector-specific primer pairs for optimal annealing temperatures using a gradient PCR experiment with the DNA templates. Test the Benchling-recommended annealing temperatures ± 3 °C initially.
12. Once annealing temperatures have been determined, create aliquots of primer solutions, DNA templates, and necessary reagents for PCR reactions for students.
Before the experiments, provide worksheets to the students to support preparation and assess understanding (Supplemental File 4, Supplemental 5, and Supplemental 6).
Data collection on learning outcomes
NOTE: If interested in collecting learning outcome data from the Gibson Assembly module and publish the results, gain approval for research with human subjects per the institution’s policies.
1. Invite the students to participate in a learning outcome investigation at least 1 week prior to the start of the Gibson Assembly independent project.
2. Have the student participants complete the multiple-choice pre-questionnaire (Supplemental File 6) the lab meeting before the start of the independent project.

2. Student experiment A: PCR to obtain linear fragments

Obtain primers and template DNA solutions for PCR from the instructor.
Pipette 25 µL reactions including DNA polymerase MasterMix, DNA template, forward and reverse primers, and nuclease-free water according to instructions in the student manual (see Supplemental File 2).
Cycle the reaction in a thermocycler according to the student manual in Supplemental File 2. Ensure that the annealing temperature of the reaction is appropriate for the primers, and that the extension time is appropriate for the length of the desired amplicon. Refer to Supplemental File 2 and Supplemental File 3 for detailed instructions.
Store PCR reactions at 4 °C or −20 °C until the following lab period.
Analyze 5 µL of each reaction via agarose gel electrophoresis. While the gel is running, add 1 µL of DpnI restriction enzyme to each reaction that utilized plasmid DNA as a template. Incubate this mixture for 1 h at 37 °C.
NOTE: DpnI will degrade any remaining circular plasmid DNA template, making it less likely to obtain false positives in the transformation step.
Image the gel to confirm that PCR was successful and correct amplification was achieved. Purify any successful PCR reactions with a commercially available PCR purification kit. Measure the concentration (ng/µL) of purified PCR product using a microvolume spectrophotometer for use in subsequent Gibson assembly calculations.
Before the next class meeting, design the reaction recipe for Gibson Assembly (see Supplemental File 2 and Supplemental File 4).

3. Student experiment B: Gibson Assembly, transformation, and selection of clones

Pipette the Gibson Assembly reaction according to the designed recipe. Incubate the reaction at 50 °C for 15 min. While reactions incubate, prepare for transformation by thawing chemically competent Escherichia coli cells on ice.
Transform 2 µL of the Gibson Assembly into chemically competent cells via heat shock. Pipette 100 µL of the transformed cells on two selection plates and spread with sterilized beads. Prepare 10^-4, 10^-5, and 10^-6 dilutions of the remaining cells on Luria-Bertani (LB) agar. Plate 100 µL of each serial dilution on LB (no selection) and spread with sterilized beads.
NOTE: This step allows for the calculation of transformation efficiency.
Incubate the plates overnight at 37 °C. Store the plates at 4 °C until the next class period.
Count colonies on all plates and calculate transformation efficiency as instructed in Supplemental File 2. Re-streak one selective plate with four colonies from either selective plate and incubate this plate at 37 °C overnight.
NOTE: This serves as a backup culture of your positive clones.
Using a marker, select, circle, and label four distinct colonies on the selection plate with the student’s initials and a number (e.g., ABC1). Retrieve a new LB-agar plate containing antibiotic for selection, use a marker to divide the plate into quadrants (i.e., fourths), and then use ~1/2 of each colony to re-streak onto the respectively labeled quadrant and the other 1/2 to inoculate a 5 mL liquid LB culture for each of the four colonies selected. Be sure to label the tubes with the respective colony identity (e.g., ABC1) and add the correct concentration of antibiotic for selection. Incubate liquid cultures in a shaking incubator at 37 °C overnight and the agar plate in a static incubator at 37 °C; store liquid cultures and plates at 4 °C until the following class period.

4. Student experiment C: Plasmid isolation and screening

Isolate plasmid DNA from the liquid cultures from Experiment B using a miniprep kit. Measure the concentration of isolated plasmid (in ng/µL).
Design a restriction digest or PCR screen to analyze the isolated plasmids (see detailed instructions in Supplemental File 2 and report expectations in Supplemental File 5).
Pipette the restriction digest reactions or reactions according to designed recipes and incubate at temperatures and durations designed earlier.
Analyze results via agarose gel electrophoresis.

5. Assessment

Have the student participants complete the multiple-choice post-questionnaire (Supplemental File 7) the lab meeting after the Gibson Assembly module is complete.
Combine all data from the pre- and post-questionnaire responses for analysis.
1. Examine the statistical significance of the increase in the average score of each individual content question between the questionnaires by using two-tailed paired t-tests and Cohen’s d effect size.
2. Evaluate the student confidence increase for each individual term and technique to determine the extent of change and its statistical significance.
3. Determine the impact of student background and academic factors on student learning by using normalized learning gain (NLG) and unpaired t-tests.
4. Assess the students’ attitudes towards the project experience and molecular biology careers by evaluating the average scores.

Representative Results

Student success in cloning
In each iteration of the Gibson Assembly module (2019, 2022, and 2023), students were asked to prepare a report summarizing their findings. In 2019, 36 of 44 students (81.8%) reported that they successfully cloned their plasmids based on the results of the screen they designed for Experiment C. A total of 14 out of 20 students (70.0%) reported success in cloning their assigned constructs in 2022, while the team-based project in 2023 had 12 of 27 students (44.4% or 4 out of 8 groups) report success based on their screening analyses. The conclusions within the student reports were reviewed by the instructor or student researchers; student interpretations were corrected if it was found that the screening results did not support their claims. One sample of each distinct plasmid was analyzed by Sanger sequencing, confirming the sequences of the plasmid inserts for all 15 desired plasmids in 2019 and all 12 plasmids in 2022. In 2023, whole-plasmid Oxford nanopore sequencing was used to analyze all 8 plasmids; however, only 1 of the 8 plasmids matched the desired sequence.

Content-based assessment of molecular biology concepts
The content section of the questionnaire comprised 10 multiple choice questions that tested students’ understanding of molecular biology and molecular cloning concepts. Comparison of the average pre- and post-questionnaire content scores for all 10 content questions revealed a significant increase from 63.7% to 80.4% (p < 0.05, two-sided paired t-test; Figure 4), suggesting that the Gibson Assembly independent project improved students’ understanding of molecular biology and cloning concepts. A Cohen’s d test determined that this significant increase corresponds to a large effect size (d = 0.891), supporting a strong difference between students’ pre- and post-responses for content questions.

Figure 4: Improvement of students’ understanding of molecular biology and molecular cloning following the independent project. Box and whisker plot of student responses to questions testing content in molecular biology and molecular cloning before and after completion of the independent project (sample size, n, of 46; Gibson Assembly Pre- and Post-Questionnaires in Supplemental File 6 and Supplemental File 7, respectively). Average pre- and post-scores were analyzed via a two-tailed paired t-test, which indicates a significant increase in correct student responses (p < 0.5). The boxes represent the middle 50% of scores (i.e., 25^th to 75^th percentiles), and the whiskers represent the minimum and maximum scores (%). Please click here to view a larger version of this figure.

Next, we examined whether the increase in the average score of each individual content question was statistically significant between the pre- and post-questionnaires. Analyses using two-tailed paired t-tests showed that student responses to questions on PCR reactions, DpnI restriction digests, Gibson Assembly enzymes, and Gibson Assembly insert and vector sequences had significant increases (p < 0.05) after completing the independent project. These content-based questions had relatively low pre-questionnaire scores compared to the overall content average of 63.7%, with the lowest scores on questions about DpnI digests and Gibson Assembly enzymes (34.8% and 47.8%, respectively). The post-project average for the aforementioned questions increased to 71.7% and 89.1%, respectively, which is closer to the overall content average of 80.4%. These data show that by the end of the project, students’ understanding of both DpnI digests and Gibson Assembly enzymes improved with a greater effect size than the overall content section of the questionnaire (d = 0.982).

Attitudinal assessment of molecular biology terms and lab techniques
The attitudinal questions were split into two sections in the student questionnaire. The first section had students self-assess their knowledge of seven molecular biology terms on a scale of 1–4 (1 = “I have no idea what this term means” to 4 = “I know what this term means and could easily explain it to someone else”). The average term confidence significantly increased from 3.52 to 3.87 with a large effect size (p = 5.32 × 10^-15 and d = 1.40, respectively; Figure 5A). The second attitudinal section asked students if they agreed with the statement of “I am comfortable performing…” eleven various molecular biology lab techniques on a scale of 1–5 (1 = “Strongly Disagree” to 5 = “Strongly Agree”). The average student confidence of these eleven techniques increased significantly from 3.82 to 4.33 with a large effect size (p = 2.08 × 10^-11 and d = 1.09; Figure 5B).

Figure 5: Increased confidence of students in their understanding of molecular biology and cloning terms and techniques following the independent project. Box and whisker plots of student confidence in molecular biology (A) terms and (B) techniques in the pre- and post-questionnaires (Gibson Assembly Pre- and Post-Questionnaires in Supplemental File 6 and Supplemental File 7, respectively). For term confidence (panel A), students selected confidence on a 4-part scale (1-“I have no idea what this term means” to 4-“I know what this term means and could easily explain it to someone else”; sample size n of 45). Lab technique confidence (panel B) was assessed on a 5-part scale (1-Strongly Disagree to 5-Strongly Agree; sample size n of 42). Average pre- and post-scores were analyzed via two-tailed paired t-tests, which indicates a significant increase in student confidence for both the term and technique datasets (p < 0.5). The box shows the middle 50% (i.e., 25^th to 75^th percentiles) of answers with whiskers showing minimum and maximum subjective confidence rankings. Please click here to view a larger version of this figure.

We evaluated the student confidence increase for each individual term and technique and determined that every term except DNA polymerase and transformation showed a statistically significant increase (p < 0.05, two-sided paired t-test; Table 1). Every student except one marked “I know what this term means and could easily explain it to someone else” for the term DNA Polymerase on both the pre- and post-questionnaires, so there was no room for improvement in that term (Table 1). For the Transformation term, 73.91% of students selected the highest confidence level (number 4 on the scale) in the pre-survey compared to 80.4% in the post-survey. Nine of the eleven lab techniques assessed showed a significant increase in student confidence; this excluded molarity calculations and gel electrophoresis (Table 1). On average, students reported high pre-questionnaire scores for both of these techniques (4.71 and 4.57, respectively, versus a technique average of 3.81), indicating students were already confident with these terms prior to the Gibson Assembly project. Questions that assessed student confidence on general biology questions (e.g., DNA polymerase, gel electrophoresis, molarity calculations) generally did not show statistically significant changes (Table 1). However, all specialized Gibson Assembly questions showed significant increases with the largest effect sizes (d > 1.6) with the technique ‘Gibson Assembly’ showing the largest change in student confidence (Table 1 and Figure 6).

Type of Content	Term/Technique	Avg Questionnare Response		Statistical Significance		Effect Size (d)
Type of Content	Term/Technique	Pre	Post	p value	Yes/No	Effect Size (d)
General Biochemistry	DNA Polymerase Term	3.98	3.98	undefined	No	0
	Molarity Calculations Technique	4.71	4.77	4.45E-01	No	0.130
	Gel Electrophoresis Technique	4.57	4.68	1.68E-01	No	0.254
Molecular Cloning	Designing Primers Technique	3.05	3.57	7.23E-04	Yes	0.536
	In silico cloning Technique	3.61	4.07	5.75E-04	Yes	0.533
	Blue-white screening Term	3.59	4.34	4.10E-06	Yes	0.655
	PCR Screening Term	3.78	4.31	1.02E-06	Yes	0.936
	Gibson Assembly Term	2.87	3.78	7.41E-13	Yes	1.69
	Gibson Assembly Technique	2.59	4.20	1.14E-15	Yes	1.61

Table 1: Significant increases with respect to attitudinal questions focused on molecular cloning, but not general biochemistry. The average on the pre- and post-questionnaire (Gibson Assembly Pre- and Post-Questionnaires in Supplemental File 6 and Supplemental File 7, respectively) for select term and technique questions. Two-tailed paired t-tests were used to assess significance (if p < 0.05, yes; if p ≥ 0.05, no). For the term DNA polymerase, student responses were identical between the pre- and post-questionnaire, thus p is undefined. The effect size of comparing student responses for each pre- and post-question was determined using Cohen’s d values (d ≥ 0.8 is considered a large effect). The larger the effect size, the higher the likelihood that the pre- and post-responses are distinct within the sample set. The sample size n is 45 and 42 for term and techniques questions, respectively. Abbreviation: AVG = average.

Figure 6: Increases for attitudinal questions about Gibson Assembly. Number of responses for student reported confidence in the Gibson Assembly (A) term and (B) technique in the pre- and post-questionnaires. Term confidence answers were on a 4-part scale (1-“I have no idea what this term means” to 4-“I know what this term means and could easily explain it to someone else”). Lab technique confidence answers ranged from 1-Strongly Disagree to 5-Strongly Agree. Please click here to view a larger version of this figure.

We next assessed whether student background factors impact how much a student learned from the independent project. Both achievement perception¹⁹ and performance²⁰ gaps have been shown to persist in undergraduate STEM courses due to gender; thus, we used normalized learning gain (NLG) and unpaired t-tests to determine whether a similar gender gap was observed in these independent projects. Based on these statistical tests, we did not find that student gender had an impact on students’ learning outcomes. We also checked whether academic factors such as major, GPA, grades in previous course work, and the final grade in BIO/CHEM 475 had a significant link to student learning outcomes from the independent project. While students who majored in biochemistry (Supplemental Table S1 and Supplemental Table S2) had higher GPAs and final course grades and each individually showed higher average NLG, the unpaired t-tests showed no statistical significance between these individual background factors and learning outcomes. Unpaired t-tests were also used to assess differences in NLG between the 2022 and 2023 cohorts. The 2023 cohort was found to have a significantly higher learning gain than the 2022 cohort for the content questions (Supplemental Table S3). This indicates that despite the lower cloning success rate of the 2023 cohort, they gained a greater understanding of molecular biology and molecular cloning concepts than the 2022 cohort. While the reasons for this difference are unclear, these data show that cloning success is not necessary for effective education with this module.

In addition to evaluating students' mastery of lab techniques, we assessed their attitudes toward the project experience and molecular biology careers. One question, featured in both the pre- and post-project assessments, gauged their comfort level with pursuing careers involving molecular biology techniques. The results reveal that the project significantly improved students' comfort in molecular biology careers (p = 8.46 × 10^-5), with the average score increasing from 3.60 to 4.09 on our 1–5 scale. Furthermore, the post-project questionnaire included inquiries about students' dedication to maximizing their learning experience and their assessment of the project’s value. Respondents rated their commitment at an average of 4.48 and considered the project a highly valuable learning opportunity, with an average assessment score of 4.73. Taken together, these findings underscore the value of the independent project in students’ academic growth as well as enthusiasm and confidence in molecular biology endeavors.

Supplemental Table S1: Anonymized raw data. All students’ answers and majors from the pre- and post-questionnaires that were collected during the 2022 and 2023 iterations of the Gibson Assembly CURE module. Reference Supplemental File 6 and Supplemental File 7 to see the corresponding pre- and post-questions, respectively. Please click here to download this table.

Supplemental Table S2: Differences in normalized learning gain by major for each section of the questionnaire. Unpaired t-tests determined that no question type showed significant differences in normalized learning gain (NLG) by major (p < 0.05). Please click here to download this table.

Supplemental Table S3: Difference in normalized learning gain by cohort for each section of the questionnaire. Unpaired t-tests showed that the 2023 cohort has a significantly higher normalized learning gain (NLG) for content questions compared to the 2022 cohort (p < 0.05). Please click here to download this table.

Supplemental File 1: Instructor Manual for Gibson Assembly CURE Module. A guide for instructors to implement the Gibson Assembly CURE Module. Step by step instructions are provided for instructors to prepare for the course by designing and screening Gibson Assembly Primers based on their template of interest. The topics and activities instructors should cover during each lecture and lab period for Experiments A–C are outlined. Please click here to download this file.

Supplemental File 2: Student Manual for Gibson Assembly CURE Module. A manual to provide to students, including relevant background information on Gibson Assembly and step-by-step protocols for each Experiment (A–C). Please click here to download this file.

Supplemental File 3: Experiment A Planning Worksheets (Individual and Team). Individual and team worksheets for students to understand and prepare for PCR amplification for Gibson Assembly fragment(s). Only one of these worksheets should be assigned by the instructor based on the structure of the independent project (individual or team-based). Please click here to download this file.

Supplemental File 4: Experiment B Planning Worksheet. Student worksheet to aid in the calculations required for the setup of the Gibson Assembly reaction. The number of fragments in the worksheet may be adjusted based on the cloning design for the plasmids of interest. Please click here to download this file.

Supplemental File 5: Experiment C Planning Worksheet. Student worksheet to aid in the design of a restriction digest or PCR experiment to screen for the plasmid of interest. Please click here to download this file.

Supplemental File 6: Gibson Assembly Pre-Questionnaire. Twenty-eight question questionnaire given to students prior to the start of the Gibson Assembly CURE Module. Please click here to download this file.

Supplemental File 7: Gibson Assembly Post-Questionnaire. Thirty question questionnaire given to students after completing the Gibson Assembly CURE Module. Please click here to download this file.

Supplemental File 8: Zip folder containing GenBank (.gb) sequence files of a polyketide hydroxylase gene, pUC19 vector, and Gibson-assembled plasmid, and Table of representative primers. Reference these files for exemplary primer design in the Benchling Assembly Wizard and completion of experimental planning worksheets. Please click here to download this file.

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, tubes of template DNA samples were labeled with very closely related names; it is possible that there were mix-ups of the DNA templates prepared by student researchers, leading to the combination of non-compatible linear fragments during the Gibson Assembly reaction performed by students in the teaching laboratory. In future iterations of this project, template DNA should be first sequenced or screened with a restriction digest to confirm the correct identity before being provided to students. Second, the 2023 iteration of this project consisted of four-fragment assemblies (one vector and three inserts), as opposed to two-fragment assemblies in previous years (one vector and one insert). This may have also lowered cloning success for this project as more fragments in Gibson Assembly can lower reaction efficiency²². In future iterations of this project, more redundancy can be built in by having more groups clone the same plasmid or having groups set up multiple Gibson Assembly reactions at various insert-to-vector ratios to increase the likelihood of successful plasmid ligation and transformation. Additionally, including longer overlaps between fragments and increasing incubation time of the Gibson Assembly reaction may increase success in assemblies with 4–6 fragments²³.

In addition to cloning novel plasmids, pre- and post-project survey results indicate that students improved their understanding of molecular biology and molecular cloning concepts, with the highest effect size for questions relating to Gibson Assembly cloning. Additionally, self-reported student confidence in molecular biology terms and laboratory techniques improved significantly after completing the project for the majority of those assessed. Gibson Assembly is an important and versatile molecular cloning technique used in academic research and in the biotechnology industry². By teaching these concepts and hands-on skills in a laboratory course, all undergraduate students have equal opportunity to graduate with research competency and hirable skills as they enter the workforce or continued education.

The Gibson Assembly curriculum described includes the five components of a successful CURE⁴. Students used scientific practices by designing their own protocols, including goals and expectations based on PCR amplicon sizes and restriction digest cuts (Supplemental File 3, Supplemental File 4, and Supplemental File 5). The independent project promotes student collaboration by dividing up the preparation of each required fragment amongst the members within a group and ultimately utilizing the fragments from each group member in one ligation reaction (Experiment A Planning Worksheet (Team-Based) in Supplemental File 3). The plasmids these independent projects aimed to clone are novel expression plasmids with relevance to research projects outside of the classroom, potentially providing data for publishable research. Similar to a more traditional research experience, cloning success rates in the CURE course were not 100%. Students were asked to evaluate variables that may have yielded negative results and to propose hypotheses explaining why the plasmid was not successfully cloned, which serves as an authentic learning experience for students regarding research outcomes.

In this Gibson Assembly CURE iteration, students cloned plasmids pertinent to Actinomycetota natural product research; however, the workflow was designed to be highly adaptable to other projects as well. The protocols presented here are generalized, allowing students to clone plasmids that could be used for a multitude of research purposes within six laboratory meetings and two lectures. The framework allows students to clone novel plasmids individually, or for students to work together to clone a single plasmid from >2 linear fragments in a team-based approach. Overall, this approach can be tailored to fit a broad range of research projects that may require varying utilization of Gibson Assembly.

This study evaluated student learning gains through a CURE-based approach to teaching Gibson Assembly, but additional insight could be gained through expansion of the study. While the pre- and post-questionnaire format allowed evaluation of learning outcomes due to the independent project, no comparison to lecture-based teaching of Gibson Assembly was performed. A comparison of the experiment-based CURE to a lecture-based or a more traditional laboratory activity, where the experimental outcomes are already designed for students, would provide more insight if improved Gibson Assembly learning outcomes are specific to the CURE course model. Lastly, while some content questions relating to Gibson Assembly showed significant improvements in understanding, others did not. Questions relating to the homologous overlaps used in Gibson Assembly, how they are installed, and how they recombine in a Gibson Assembly cloning reaction did not improve. This suggests that further explanation or an additional learning assessment on these topics could improve student understanding in future implementations of this curriculum.

The Gibson Assembly independent project structure continues to be implemented at Cal Poly. In future iterations of the course, this project will be modified to allow for higher student independence, hypothesis generation, and experimental design. An upcoming curriculum shift at Cal Poly presents the opportunity to lengthen the independent project to include student-led primer design, vector backbone selection, sequence analysis, and/or downstream testing of plasmids, such as for protein expression. Additionally, the independent project could be customized to teach other relevant molecular biology techniques such as site-directed mutagenesis or gene editing depending on the needs of ongoing research projects. Altering the techniques students utilize in the independent project could provide more discernment on which topics are appropriate for the CURE format, and which prove difficult to implement.

Taken together, this report details an adaptable Gibson Assembly teaching module that implements a CURE to both improve student understanding of an emergent cloning methodology and to increase competency in the scientific process. The independent project improved student learning outcomes and confidence in the majority of the molecular biology terms and techniques assessed. We encourage the use and adaptation of this module to not only create new research plasmids, but also to improve student accessibility to and confidence in research techniques, better preparing undergraduates for future careers in STEM. Ideally, this work will inspire improvements in molecular biology curricula and encourage STEM course adaptability as scientific research methodologies continue to evolve.

Divulgations

The authors have nothing to disclose.

Acknowledgements

Authors gratefully acknowledge Andrea Laubscher for technical support, and Michael Black, Sandi Clement, and Javin Oza for helpful discussions on teaching lab implementation and assessment of learning outcomes. Authors appreciatively acknowledge all students who participated in the learning outcome study in the 2019, 2022, and 2023 cohorts, as well as research students Nathan Kuhn and Aayushi Adettiwar who assisted in reagent preparation for teaching lab implementation. Authors also acknowledge funding support from the William and Linda Frost Fund, Center for Applications in Biotechnology’s Chevron Biotechnology Applied Research Endowment Grant, and the National Science Foundation (NSF-1708919 and NSF-2300890).

Materials

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

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