Undergraduate Biochemistry Laboratory Research Articles

In Vitro Characterization of Dopaminergic Toxins: A Laboratory Exercise Suitable for a Senior Undergraduate Biochemistry Laboratory Curriculum

<i>In Vitro</i> Characterization of Dopaminergic Toxins: A Laboratory Exercise Suitable for a Senior Undergraduate Biochemistry Laboratory Curriculum

Comparing Learning Outcomes and Student and Instructor Perceptions of a Simultaneous Online versus In-Person Biochemistry Laboratory Course.

This article compares the learning outcomes and student perceptions of a one semester undergraduate biochemistry laboratory course that was taught using either a fully online or a fully in-person teaching modality. The semester long biochemistry laboratory mimicked the work sequence a researcher would encounter when transforming a plasmid containing a gene for a recombinant protein (superfolder green fluorescent protein, sf-GFP) and then purifying, identifying, and characterizing that protein. The two modalities of the course were completed in the same semester, by the same instructor, in which students self-selected into which modality they preferred at the beginning of the semester. Students in the in-person section reported enjoying the laboratory course more than the online cohort of students and found it to be less time-consuming. Additionally, a survey of biochemistry laboratory instructors from across the United States, who had experience teaching both online and in-person biochemistry laboratories, indicated that the majority of instructors that responded to the survey preferred the in-person modality: believing them to be more effective and engaging for the students, more enjoyable, and less time-consuming for the instructor. Statistical analysis of formative and summative assessments indicated no significant difference in non-hands-on student learning objective and learning goal scores between the two groups, but the small number of students and instructors in this study limits the generalizability of these results.

An improved lysozyme substrate preparation method for the biochemistry kinetics laboratory

The mutation and growth of Bacteriophage T4 Lysozyme (T4L) is easily accomplished in an undergraduate biochemistry laboratory and allows students to visualize how mutations affect enzyme stability and kinetics. However, full Michaelis-Menten parameters have been difficult to observe due to lack of a readily available, small molecule substrate. A FITC-tagged peptidoglycan has been used previously, but it is not commercially available and its preparation method was not appropriate for the undergraduate laboratory. Here, we describe a cost-effective, non-toxic, and reproducible means of generating FITC-tagged peptidoglycan fragments. Fluorescence assays monitoring substrate digestion using HEWL and T4L produced signals sufficient for reliable analysis of lysozyme kinetics.

The BASIL cure: Using structure to predict function in protein biochemistry.

We have developed an undergraduate biochemistry lab curriculum based on authentic inquiry. This course-based undergraduate research experience (CURE) allows a large number of students to gain skills often learned only in the traditional one-on-one mentor-student research model by bringing a research project into the undergraduate teaching lab. The Biochemistry Authentic Scientific Inquiry Lab (BASIL) uses a combined computational and wet lab approach to study proteins of known structure but unknown function. Over 3800 structures in the Protein Data Bank (PDB) have unknown function. Students following the BASIL curriculum use a combination of sequence and structure alignment tools to study these structures with the goal of identifying possible enzymes. They then use molecular docking to predict what model substrates fit near a proposed active site. Students can produce the target enzymes in the lab using standard wet-lab biochemistry techniques for expression and purification, and they then perform kinetic assays with model substrates selected from their docking studies. We have successfully used this curriculum in biochemistry lab courses for majors and non-majors. The curriculum is modular and can be used as a whole or individual parts may be incorporated into an existing course - either lecture or lab. Curriculum materials are available free-of-charge at basilbiochem.org. We are currently investigating how this curriculum can be used in a variety of academic settings, and we welcome new collaborators. We can offer synchronous support via virtual meetings and asynchronous support via Slack. This project is supported in part by NSF IUSE 2141908.

Abstract 1496: The Exploration of Yeast Alcohol Dehydrogenase Thermal Stability in the Undergraduate Biochemistry Laboratory

Abstract 1496: The Exploration of Yeast Alcohol Dehydrogenase Thermal Stability in the Undergraduate Biochemistry Laboratory

A report on learning gains and student confidence after incorporating an enzyme purification and characterization course-based undergraduate experience.

Course-based undergraduate research experiences (CUREs) can provide undergraduate students access to research opportunities when student and faculty resources are limited. In addition to expanding research opportunities, CUREs may also be explored as a pedagogical tool for improving student learning of course content and laboratory skills, as well as improving meta-cognitive features such as confidence. We examined how a 6-week CURE in an upper-level undergraduate biochemistry lab affected student gains in content knowledge and confidence in scientific abilities, compared to a non-CURE section of the same course. We find that gains in content knowledge were similar between CURE and non-CURE sections, indicating the CURE does not negatively impact student learning. The CURE was associated with a statistically significant gain in student confidence, compared to non-CURE group. These results show that even a relatively short CURE can be effective in improving student confidence at scientific research skills, in addition to expanding access to research.

From classroom to clinic: Biochemistry lab for pre-health majors.

Undergraduate laboratory courses are vital for both enhancing student learning and preparing students for their future careers. Despite their importance, laboratory courses are often met with a lack of enthusiasm from students. For pre-health students specifically, laboratory courses are commonly seen as part of a check-list that needs to be completed in order to achieve future education or a career, instead of seeing the information that is taught in laboratories as essential preparation. A one-semester biochemistry laboratory module was designed to demonstrate the real-life applications of experimental techniques. This laboratory module introduced students to common clinical measurements and included learning metabolite analysis through hands-on experiments, connections to simulated patient visits, generation of a research question, and implementation of a student-designed independent experiment. Surveys indicate that this approach was helpful in creating a greater understanding of the applicability of undergraduate laboratory concepts appealing specifically to pre-health students. Additionally, students found this module to provide a variety of gained benefits, knowledge, and confidence in performing scientific techniques. The outcomes of this laboratory module indicate its success and potential to be used in curricula as an effective way to engage pre-health students in an undergraduate biochemistry laboratory.

Mushroom Tyrosinase Assay is used to Teach Enzyme Kinetics and Inhibition Studies in an Undergraduate Biochemistry Course

A spectrophotometric mushroom tyrosinase assay was optimized for use in the undergraduate biochemistry laboratory course at Virginia Tech. The laboratory exercise is designed to teach students about enzyme kinetics and tyrosinase‐inhibitor interactions. The tyrosinase protein structure was first studied using the python based molecular visualization software PyMOL and a PDB ID from the Protein Data Bank. The PDB ID that was used was PY9W and displays a H(2) L(2) tetramer. The H subunits of the complex contain a copper binding site that is coordinated by 3 histidine residues. The structure/function relationship was further studied using an immersive modeling hypercube. The assay uses white button mushrooms as the source of the tyrosinase enzyme, l‐3,4 dihydroxyphenylalanine (L‐DOPA) as the substrate, and kojic acid as the inhibitor. The optimal amounts of enzyme, substrate, and inhibitor are first determined, then a series of experiments are conducted which test varying concentrations of L‐DOPA against varying concentrations of kojic acid. Change in absorbance is measured at 475 nm in a spectrophotometer and the data is plotted to determine the kinetic constants and type of inhibition. Experimental kinetic constants are then compared to previously reported values. Further protein concentration in the tyrosinase extract is determined using Bradford assays. The incorporation of this exercise into the undergraduate biochemistry laboratory course at Virginia Tech will allow students to explore enzyme kinetics and tyrosinase‐inhibitor interactions.

Characterizing Novel Kinases as an Authentic Research Experience in the Undergraduate Biochemistry Lab

The protein databank (PDB) contains 183,000 structures, with approximately 4200 of those currently classified as having “unknown function”. Based on structures with known function(s) in the PDB, about 2500 of those proteins are expected to be enzymes. The Biochemistry Authentic Scientific Inquiry Lab (BASIL) consortium has developed a series of modules that guide undergraduate students through in silicoprediction of the function of proteins of “unknown” function, along with modules for in vitro confirmation of these predictions. We used these modules as part of a kinase‐focused course‐based undergraduate research experience (CURE). Kinases were selected because of their abundance in humans (over 500 members of the kinase superfamily) and their involvement in almost every aspect of cellular function. Multiple putative kinases were characterized by undergraduates, using the in silicoBASIL modules (which utilize BLASTp, Pfam, SPRITE and Dali). Specific potential substrates were then identified using SwissDock and/or AutoDock Vina (via PyRx). Students were able to successfully overexpress the putative kinases and subsequently purify them using affinity chromatography. Coupled in vitro kinase assays were performed first using the well‐characterized Abl kinase and an appropriate peptide substrate. Students then adapted the coupled kinase assay to evaluate the potential substrates they had identified. Students reported that having an integrated laboratory experience, with the ability to make their own decisions based on their in silicodata, gave them a strong sense of ownership of their projects. Additionally, the techniques they learned were all used to support their hypotheses, which helped them understand the details of why and how each method was performed. Overall, they described it as a positive experience and one of their more interesting laboratory courses.

Using structure to predict function in protein biochemistry: a combined computational and wet lab approach

We have developed an undergraduate biochemistry lab curriculum based on authentic inquiry. The Biochemistry Authentic Scientific Inquiry Lab (BASIL) uses a combined computational and wet lab approach to study proteins of known structure but unknown function. There are over 3800 structures in the Protein Data Bank (PDB) that have unknown function. Students use a combination of sequence and structure alignment tools to study these structures with the goal of identifying possible enzymes. They then use molecular docking to predict what model substrates fit near a proposed active site.

An Approach to Transitioning Undergraduate Biochemistry Laboratory Courses Online

An Approach to Transitioning Undergraduate Biochemistry Laboratory Courses Online

Design of a Robust Undergraduate Biochemistry Laboratory Module Based on a Purification Scheme for Bovine Serum Albumin

This paper describes an undergraduate biochemistry laboratory module consisting of a set of experiments designed around a purification scheme for bovine serum albumin (BSA). Students purify BSA from cow plasma by a combination of salt and acetone precipitation, equilibrium dialysis, ion exchange, and size exclusion chromatography. Students use the bromocresol green–BSA complex assay to quantify albumin at each step in the scheme and generate a purification table. In addition, trace amounts of IgG’s and nucleases, the major contaminants in BSA purification, are probed by Western blotting and DNase assay, respectively. This module exposes students to the principles and techniques of separation of proteins based on solubility, size, and surface charge. In addition, students learn the concept of limit of detection of analytical techniques such as SDS-PAGE and Western blotting. The module utilizes two 3-h laboratory periods, with a 1-h prelab lecture per week, for about 6 weeks. Depending on class size, groups consist of two to four students. The module is suitable for students who have completed a semester of biochemistry. Instructors can extend the module to advanced level laboratory by including enzyme kinetics and protein structure–function studies based on albumin’s pseudoesterase activity and intrinsic tryptophan fluorescence, respectively.

Student design and characterization of visible DHFR fusions for biochemistry tools to improve learning during lab exercises

AbstractStudent feedback from an undergraduate biochemistry lab course suggested the use of visibly traceable proteins may assist learning. Based on this feedback, we used guided inquiry lab exercises where students developed and characterized a suite of fluorescent protein‐dihydrofolate reductase (DHFR) fusions as tools for a biochemistry teaching lab. In contrast to the unfused versions, members of this suite are well‐expressed, soluble, visible, highly stable, and easily characterized. The color of mCherry and EGFP fluorescent fusions with microbial DHFR allows students to visibly track their target protein from expression through purification under ambient light, while fusions with BFP are visible under UV‐light. Fusions were made to both wild‐type and kinetically enhanced DHFR variants. Importantly, we found that fluorescent protein fusions with DHFR did not kinetically interfere as the KM and kcat values were not remarkably altered from the unfused variant. With these fusions, students can easily measure kinetic parameters under steady‐state conditions with readily available substrate and common laboratory spectrophotometers. Additionally, students also determined IC50 values of trimethoprim for DHFR. These exercises can be completed in a series of up to six lab periods and we have included the protocols for instructors who wish undertake a similar series of experiments in their biochemistry teaching labs. Using these visible fusion enzymes with subsequent students, we observed potential learning gains on a course assessment and received positive student feedback. We suggest that the often over‐looked element of visual cues in a biochemistry lab may be an exploitable component of learning.

Analysis of Amylase in the Kitchen: An At-Home Biochemistry Experiment for the COVID-19 Pandemic

During the COVID-19 pandemic, an at-home enzyme assay was developed for a biochemistry laboratory course at Arizona State University. The experiment was designed to use items that could be easily obtained and safe to use. The experiment focused on the analysis of salivary amylase using starch from food as a substrate, black tea as an inhibitor, and tincture of iodine to quantify the amount of starch present. In this article, we describe the design of the experiment and an analysis of the student performance with specific mention of the common problems that the students experienced. While this experiment was specifically designed as an at-home experiment, it could easily be adapted to use in a typical undergraduate biochemistry laboratory.

Microsoft OneNote provides continuity for undergraduate biochemistry lab during a pandemic.

This article illustrates the use of Microsoft OneNote as an Electronic Notebook for undergraduate biochemistry lab reports as well as student opinions of this use both before and during the online coursework shift during the pandemic.

Implementation of research on potential drug target cloning and characterization in a biochemistry laboratory.

An approach for incorporating preliminary drug discovery research into a biochemistry laboratory is described. During a total of 42 hr (one 3-hr laboratory section a week for 14 weeks), students were exposed to bioinformatics; molecular cloning; protein expression, purification, and characterization; and enzymatic kinetic assays. This research-oriented laboratory not only includes the standard elements of common undergraduate biochemistry laboratory manuals but also emphasizes the logical connections among biochemistry laboratory techniques. Moreover, this approach exposed students to laboratory research and the concept of drug discovery.

A guided-inquiry investigation of bacterial membrane potential using flow cytometry for an undergraduate biochemistry laboratory course.

Flow cytometry has become an important tool in the life sciences and medical fields, yet there are often few opportunities for undergraduate students to receive training with this type of instrumentation as part of life science curricula at many colleges and universities. We describe a straightforward laboratory exercise designed for a college-level biochemistry course that uses flow cytometry to investigate changes in bacterial membrane potential and cell viability in response to various treatments. Anecdotally, we have noticed that many students often have difficulty understanding the concept of membrane potential and the essential role that it plays in cellular processes. Therefore, this exercise also provides a pedagogical tool for visualizing changes in cellular membrane potential as way to enhancing students' comprehension of this abstract concept. Students' understanding of flow cytometry and membrane potential was assessed using a preactivity and postactivity quiz; the results indicate that the students' understanding of these concepts significantly improved after they completed this laboratory exercise. © 2019 International Union of Biochemistry and Molecular Biology, 48(1):61-66, 2020.

Supporting Learning in the Undergraduate Biochemistry Laboratory at an Australian University Through a 3‐Phase Approach Using Technology, eNotebooks and Partnering with Students

At the University of Wollongong in Australia, Principles of Biochemistry (BIOL213) is a second year undergraduate (UG) subject/course taught to over 450 students studying a variety of degree programs. A review of this subject recommended the redesign of the laboratory section to enhance the learning experience by supporting learning outcomes and achievement of deeper learning of challenging content.To address this issue a 3‐phase approach was undertaken utilizing technology in all phases. The phased approach is a blended learning environment in which phases 1 and 3 were completed online whilst phase 2 was conducted face‐to‐face in the laboratory. Phase 1 aimed to provide an engaging online learning environment that would encourage students to learn independently and, to provide tools to help students feel better prepared and less overwhelmed for group activities encountered in class. The in‐class activities were phase 2 and utilized an electronic notebook (LabArchives) whereby students conducted experiments generating data and forming conclusions whilst collaborating in groups. The in class reports were submitted and graded through the eNotebook in groups, fostering collaboration among students. Phase 3 included post‐laboratory online quizzes that the students completed through the subject eLearning site within 3 days of the completion of the laboratory class. The aim was to test understanding and longer term retention of knowledge, whilst contextualising student understanding using a different, but related, scenario.This project included undergraduate students as partners (SAP) utilising an action research framework, where UG students who had completed the subject assisted in the design of the eNotebook and training package, the incorporation of the eNotebook into teaching, design of the evaluation tool, and dissemination of the research findings. The UG students were perfectly placed to discuss the curriculum that will underpin the eNotebook design and due to their involvement in a trial of the eNotebook they provided crucial insights into the design of the student training package.Results show over 80% of students felt the phase 1 online activities helped prepare them for the phase 2 in‐class activities and assisted their learning and over 70% felt the phase 3 online activities enhanced and reinforced their learning. However the preference from students for an electronic versus paper notebook were mixed, although over 70% of students agreed that the group discussion and collaboration assisted their learning. A comparison in assessment performance by student groups pre‐ and post‐initiative shows an improvement in performance in laboratory‐related activities. This session will present the triumphs and challenges of implementing this approach with some points to consider for those wanting to pursue similar initiatives and wanting to partner with students in curriculum design.Support or Funding InformationUniversity of Wollongong Educational Strategies Development FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Undergraduate laboratory experiment on determination of total protein content in milk powder by moving reaction boundary titration.

Laboratory exercises focused on protein quantification are frequently conducted in traditional undergraduate biochemistry laboratory curriculum. The laboratory course described here is designed to provide students with experience in measurement of protein content in milk powder by moving reaction boundary titration (MRBT), a new rapid technique for total protein content determination in milk. In addition, this approach is weakly influenced by nonprotein nitrogen reagents such as melamine and urea. The course was done as three weekly laboratory exercises. First, students established a standard curve for milk protein concentration by MRBT method. Then, students investigated the influence of nonprotein nitrogen reagents on MRBT method. Finally, students made a comparison among three different protein quantification methods (MRBT, Biuret, and Kjeldahl method). From the experiments, students grasped the concept and advantages of MRBT and deepened the understanding of protein quantification. This course offer students the opportunity to be exposed to an advanced technique, which may have practical significance to their future study and work in the life science field. © 2018 International Union of Biochemistry and Molecular Biology, 46(6):644-651, 2018.

Genotype and Phenotype of Caffeine Metabolism: A Biochemistry Laboratory Experiment

An experiment for the upper-division undergraduate biochemistry laboratory is described in which students investigate the influence of genetic variations of cytochrome P450 1A2 on drug metabolism, using caffeine as a model compound. Saliva samples from human subjects are characterized for a single-nucleotide polymorphism in the CYP1A2 gene (genotyping) and for the rate of caffeine clearance (phenotyping). This experiment has clinical significance in the area of personalized medicine.