Undergraduate Biochemistry Course Research Articles

Improvement of student understanding of how kinetic data facilitates the determination of amino acid catalytic function through an alkaline phosphatase structure/mechanism bioinformatics exercise

Laboratory exercises, which utilize alkaline phosphatase as a model enzyme, have been developed and used extensively in undergraduate biochemistry courses to illustrate enzyme steady-state kinetics. A bioinformatics laboratory exercise for the biochemistry laboratory, which complements the traditional alkaline phosphatase kinetics exercise, was developed and implemented. In this exercise, students examine the structure of alkaline phosphatase using the free, on-line bioinformatics protein-modeling program Protein Explorer. Specifically, students examine the active site residues of alkaline phosphatase and propose functions for these residues. Furthermore, by examining the mechanism of alkaline phosphatase and by using the published kinetic data, students propose specific roles for several active-site residues. Paired t-test analysis of pre- versus postexercise assessment data shows that the completion of the exercise improves student's ability to use kinetic data correctly thereby determining a probable catalytic function for an active site amino acid.

Introducing principles of validation into biochemistry and biotechnology courses

Although undergraduate biochemistry and biotechnology courses teach the concept of accuracy and precision during practical laboratory sessions, the formal teaching of validation methodologies receives little attention. An increasing number of biochemistry and biotechnology graduates are finding work in industry in the area of industrial validation associated with biopharmaceutical, diagnostics, biomedical device, and pharmaceutical validation. We have introduced a structured introduction to validation into our undergraduate industrial biochemistry programme to illustrate the importance of validation within a framework of good manufacturing practice (GMP) and to show how validation is essential for regulatory compliance.

Introductory bioinformatics exercises utilizing hemoglobin and chymotrypsin to reinforce the protein sequence‐structure–function relationship

We describe two bioinformatics exercises intended for use in a computer laboratory setting in an upper-level undergraduate biochemistry course. To introduce students to bioinformatics, the exercises incorporate several commonly used bioinformatics tools, including BLAST, that are freely available online. The exercises build upon the students' background knowledge of hemoglobin and chymotrypsin, and foster a better understanding of how protein sequence relates to structure and function.

Reversible ligand binding reactions: Why do biochemistry students have trouble connecting the dots?

Adaptive chemical behavior is essential for an organism's function and survival, and it is no surprise that biological systems are capable of responding both rapidly and selectively to chemical changes in the environment. To elucidate an organism's biochemistry, its chemical reactions need to be characterized in ways that reflect the normal physiology in vivo. This is a challenging experimental problem because biological systems are inherently complex with myriads of interlinked chemical networks orchestrating processes that are mostly irreversible in nature. One successful approach for simplifying the study of biochemical reactions is to analyze them under controlled reversible equilibrium conditions in vitro that approximate the range of physiological conditions found in vivo. Because this approach has helped elucidate some of the chemical mysteries of complex biological systems, many topics presented in modern biochemistry courses are essentially rooted in the chemistry of reversible equilibrium reactions. Since most undergraduate biochemistry courses typically require students to complete year-long general and organic chemistry courses, biochemistry instructors may assume that entering students have sufficient understanding of basic reversible equilibrium chemistry to move forward into more advanced biochemical topics. However, this assumption is at odds with our experience in that many entering students seem confused by the conventions, language, symbolic formalism, and/or mathematical tools normally use to describe reversible equilibrium reactions. Part of the problem here may stem from how certain basic chemical concepts are taught (or are not taught) in their prerequisite chemistry courses. Here, we identify some conceptual barriers that many students seem to confront and we discuss instructional strategies designed to help students "connect the dots," so to speak, and better understand how dynamic biological processes can be analyzed in terms of reversible equilibrium chemistry.

Introducing Michaelis–Menten Kinetics through Simulation

We describe a computer tutorial that introduces the concept of the steady state in enzyme kinetics. The tutorial allows students to produce graphs of the concentrations of free enzyme, enzyme–substrate complex, and product versus time in order to learn about the approach to steady state. By using a range of substrate concentrations and rate constants, students are able to simulate enzyme kinetics and produce Lineweaver–Burk plots. The tutorial is intended for use in third- or fourth-year undergraduate biochemistry courses.

Spectroscopic Measurement of the Redox Potential of Cytochrome c for the Undergraduate Biochemistry Laboratory

Teaching redox chemistry is an important facet of undergraduate biochemistry courses, particularly with respect to developing the students' understanding of the central metabolic processes such as the mitochondrial respiratory chain and photosynthesis. We have introduced an experiment for the undergraduate metabolism course involving the spectroscopic measurement of the redox potential of cytochrome c. The experiment permits a quantitative result within a single three-hour laboratory period and complements the basic redox concepts and calculations shown in class.

Interactive Software for the study of membrane biology: lipid composition, solubilization and liposome reconstitution and characterization

Biological membranes define cellular boundaries, divide cells into discrete compartments, organize complex reaction sequences, and act in signal reception and energy transformations. This topic is studied in all undergraduate biochemistry courses. Visualization of structures generally facilitates the understanding of many related topics of membrane composition, structures, and protein interactions but they lack in many events that occurs in membranes. Also, at the present time, animations exploring solubilization and reconstitution of membrane proteins in vesicular systems are not available. Thus, we have developed a software named AnimaBio, in Macromedia Flash 7.0, whose principal objective was the animation of some processes used in the study of membrane biology and it was didactically divided in: (1) composition and physics properties; (2) construction of systems mimetically to natural membranes and (3) characterization of these biomimetic systems using experimental examples.The topics explained in each section were: (1) Membranes composition; lipids and proteins distribution; fluid mosaic model; the basic structural unit of lipid bilayer; peripheral proteins; anchored proteins; integral proteins; covalently attached oligosaccharides; solubilization of proteins and hemolytic effects; (2) construction of biomimetical systems using different techniques; sonication followed by direct insertion of proteins and co-solubilization methods; (3) Kinetic properties of the enzyme, reconstituted in the vesicular system, using examples of actions of different agents such as: inhibitors, detergents, ionophores and photosensitive dyes. All topics were illustrated in the animation using some examples such as: erythrocytes membranes; alkaline phosphatase (which have a GPI anchor) and integral proteins such as Na,K-ATPase. AnimaBio has many animations exploring some fragile concepts and each part was also explained in a text. Some items were enriched with glossary definitions, compound structures and technical terminology. This approach allows the student to have a non-fragmented view of membrane biology and to understand the importance of a given interaction of lipid and protein as a whole. Thus, the software provides a non-passive study facilitating the comprehension of this important topic in the biochemistry course and also motivating the student to search the literature for similar examples.

Developing Biochemistry Concept Inventories for Diagnosing Students’ Misconceptions

The Force Concept Inventory presented by Hestenes (The Physics Teacher, Vol. 30, pp. 141–158) has been widely adopted and used to reform physics education. However, the field of biochemistry is not yet served by similar assessment instruments, as pointed out by Hal White who emphasized the need for more conceptual questions in biochemistry (Biochem. Mol. Bio. Ed., Vol. 33, pp. 227–8). Our current research focuses on developing biochemistry concept inventories (BCCIs) based on a pilot study in undergraduate biochemistry courses where student responses to the distracters of the pre-assessment questions were mapped to specific known misconceptions. We will present the use of think-aloud protocols to ascertain the validity of the questions. The BCCIs will consist of three thematic sections: (1) structure and function of biomolecules, (2) properties of amino acids and (3) reversible equilibrium. When appropriately used, concept inventories have been shown to have significant effects in reforming undergraduate science education. They provide powerful summative assessments; and perhaps more importantly, they can be formative assessments that aid instructors in adjusting their pedagogical strategies to remediate the misconceptions diagnosed. Thus, development and use of BCCIs should lead to significant improvements in the way biochemical instruction is conducted in the classrooms of the future.

ABO Blood Group Genotyping Laboratory for Undergraduate Biochemistry Students

ABO blood groups are routinely discussed in an undergraduate biochemistry course to illustrate the glycosylation of proteins. In the past, an ABO blood typing laboratory using the student's own blood accompanied this discussion. With our current knowledge of bloodborne pathogens, we no longer perform student laboratories that require human blood. To circumvent this problem, we have designed a simple ABO PCR-RFLP genotyping laboratory that only requires a small amount of DNA from the student. Student DNA can easily be extracted from saliva. PCR primers amplify a region of the ABO gene that is dissimilar amongst the ABO genotypes. The PCR amplimers are then digested by restriction enzymes that differentially recognize the various ABO genotypes. DNA fragments are visualized by standard agarose gel electrophoresis and the student genotype is determined by the number and size of the resulting bands. This genotyping method is more informative than standard blood typing because it allows the student to determine both of their ABO glycosyl transferase alleles.

Development of a green fluorescent protein‐based laboratory curriculum

A laboratory curriculum has been designed for an undergraduate biochemistry course that focuses on the investigation of the green fluorescent protein (GFP). The sequence of procedures extends from analysis of the DNA sequence through PCR amplification, recombinant plasmid DNA synthesis, bacterial transformation, expression, isolation, and characterization of the protein by SDS-PAGE. A survey of participants found that the majority of them were performing most of these procedures for the first time and that participants found the exercises enjoyable and considered them a significant aid to their understanding of biochemistry, cell biology, and molecular genetics.

Determination of the Rh factor: A practical illustrating the use of the polymerase chain reaction

A practical experiment on the PCR is described that has been used over several years as part of an undergraduate biochemistry and molecular biology course for chemistry students. In the first experimental session, students prepare their own DNA samples from epithelial cells of the mouth and use them as templates in the PCR. In the second session, they analyze the amplified DNA by electrophoresis and determine their Rh factor.

Profile of Robert G. Roeder

Profile of Robert G. Roeder

DNA isolation from a dried blood sample, PCR amplification, and population analysis: Making the most of commercially available kits

AbstractA 3‐week undergraduate molecular biology laboratory exercise combining commercially available kits is described. The laboratory involves DNA extraction from a human dried blood sample, amplification of an Alu‐repeat sequence found on chromosome 8, and analysis of the genetic and allelic frequency in the class population using Hardy‐Weinberg statistics. The use of the prepared kits provides a means for the instructor of an undergraduate biochemistry course to explore molecular biology techniques on a limited budget and with a minimal time investment.

Promising New Directions in Biochemistry

Promising New Directions in Biochemistry

Different energy sources in sports: Introductory software

AbstractThis work describes software designed to promote the association between the content of a basic undergraduate biochemistry course and the professional activities of physical education students. The software contains three main content sections: (A) Structure of skeletal muscle, (B) Contraction mechanism, and (C) Adaptations to physical exercise. A fourth section asks questions of the students. The software offers students a brief introduction on muscle structure and function, focusing on the energy sources required by different kinds of physical activities. The software was field tested at Universidade Estadual de Campinas (Unicamp), Brazil and has been adopted by several Brazilian universities. The students are required to examine the software, to discuss its contents, and to produce a list of questions arising from their work with the software. These questions are answered during the development of the curriculum, thereby connecting biochemical knowledge with the energetic needs for the practice of sports.

Identification of Forensic Samples via Mitochondrial DNA in the Undergraduate Biochemistry Laboratory

A recent forensic approach for identification of unknown biological samplesis mitochondrial DNA (mtDNA) sequencing. We describe a laboratory exercisesuitable for an undergraduate biochemistry course in which the polymerasechain reaction is used to amplify a 440 base pair hypervariable region ofhuman mtDNA from a variety of "crime scene" samples (e.g., teeth, hair,nails, cigarettes, envelope flaps, toothbrushes, and chewing gum). Amplificationis verified via agarose gel electrophoresis and then samples are subjectedto cycle sequencing. Sequence alignments are made via the program CLUSTALW, allowing students to compare samples and solve the "crime."

Determination of DNA Bases Using Electrochemistry: A Discovery-Based Experiment

A discovery-based experiment is presented for use in undergraduate analytical and biochemistry courses. The experiment uses electrochemical techniques (e.g., cyclic, linear-sweep, and/or square-wave voltammetry) to detect the presence of DNA bases in solution. Working individually or in teams, students must develop a method for the detection of adenine(A), guanine (G), cytosine (C) and thymine (T) in aqueous samples. They are given only topical information about their project and must research and plan the analyses, learn the instrumental methods to be used, and prepare an experimental protocol that will be “validated” by another individual/team during a subsequent laboratory. Goals of this approach include introducing students to various electrochemical techniques and having them research how these techniques are being used to determine and study biologically relevant analytes. Another goal is to place students in the position of being scientists and having to make decisions and recommendations. Each step of the analytical process must be carefully considered and its significance assessed because there are no “recipes” to follow as they develop their methods and make comparisons between different electrochemical techniques for the determination of analytes.

Study of protein‐ligand binding by fluorescence

AbstractA practical class experiment is described to show the students an application of fluorescence spectroscopy in the study of protein‐ligand binding. This class is part of an undergraduate physical biochemistry course for life science students. The major aim is to introduce the students to the basic concepts of fluorescence emission (intrinsic and extrinsic), the effect of environment on fluorescence parameters, and how this could be applied to the study of ligand binding to proteins. In particular, binding of the fluorescent probe 1‐anilinonaphthalene‐8‐sulfonate to albumin (native and oxidized) is described. From the obtained results, the stoichiometry of complex formation, the binding equilibrium constants, and alterations of the environment of the binding sites are determined and discussed with the students.

Teaching and Assessing Three-Dimensional Molecular Literacy in Undergraduate Biochemistry

Structural concepts such as the exact arrangement of a protein in three dimensions are crucial to almost every aspect of biology and chemistry, yet most of us have not been educated in three-dimensional literacy and all of us need a great deal of help in order to perceive and to communicate structural information successfully. It is in the undergraduate biochemistry course where students learn most concepts of molecular structure pertinent to living systems. We are addressing the issue of three-dimensional structural literacy by having undergraduate students construct kinemages, which are plain text scripts derived from Protein Data Bank coordinate files that can be viewed with the program MAGE. These annotated, interactive, three-dimensional illustrations are designed to develop a molecular story and allow exploration in the world of that story. In the process, students become familiar with the structure-based scientific literature and the Protein Data Bank. Our assessment to date has shown that students...

A Novel and Innovative Biochemistry Laboratory: Crystal Growth of Hen Egg White Lysozyme

Protein crystallization is the first and perhaps most important step in solving the structure of a protein via X-ray crystallography. However, very little time, if any, is spent working on this during laboratory experiments in undergraduate biochemistry courses. To remedy this, we developed a semester-long biochemistry laboratory experience that mimics a research setting centered around protein crystallization. Students are given purified lysozyme and a range of initial conditions. It is then their task to refine the crystal growth conditions by thinking critically about the problem. By understanding the theory of protein crystallization, students can manipulate whatever variable they feel will give the best final result. Throughout the semester the students set up numerous trials, learning from their previous results, in an effort to grow good single crystals of lysozyme. After good crystals are obtained, the students are asked to reproduce their results, a task that is required in a research setting. This experiment tests both the students' ability to think about a problem and their analytical techniques.