In course evaluations, the students cited a high degree of satisfaction with the project-based approach. Keywords: Problem-based experiments, independent ...
© 2003 by The International Union of Biochemistry and Molecular Biology Printed in U.S.A.
BIOCHEMISTRY
MOLECULAR BIOLOGY EDUCATION Vol. 31, No. 5, pp. 303–307, 2003
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Articles An Undergraduate Biochemistry Laboratory Course with an Emphasis on a Research Experience* Received for publication, January 22, 2003, and in revised form, April 25, 2003 Mary Lou Caspers‡ and Elizabeth S. Roberts-Kirchhoff From the Department of Chemistry and Biochemistry, University of Detroit Mercy, Detroit, Michigan 48219-0900
In their junior or senior year, biochemistry majors at the University of Detroit Mercy are required to take a two-credit biochemistry laboratory course. Five years ago, the format of this course was changed from structured experiments to a more project-based approach. Several structured experiments were included at the beginning of the course because not all students were familiar with computer-based statistics and graphics programs or fundamental biochemical techniques. Finally, students were given an enzyme purification assignment and a test/control project. In the latter project, students were provided with tissue from test and control animals and were expected to propose and test a parameter that they hypothesized might be different between the two tissues. For both projects, student teams were required to search the literature, submit and orally defend their proposals, perform the experiments, and submit a report. For all experiments except the test/control project, students submitted a written report in the style of a journal paper. A poster presentation was required for the test/control project. In course evaluations, the students cited a high degree of satisfaction with the project-based approach. Keywords: Problem-based experiments, independent projects, experimental design, poster presentations, research reports.
Problem-solving abilities and experimental design are important skills for any scientist, but they are not taught in laboratory courses where students simply follow an explicit set of instructions to obtain results. Problem-based learning is a method in which students are presented with a goal that can be reached through different paths.1 As students work toward the goal, they acquire problemsolving, self-directed learning and team skills.1 Unless the curriculum requires an independent research project, students who do not elect undergraduate research do not have the opportunity to hone such skills in traditional, “cookbook” laboratories. Problem-based laboratories have recently been implemented in introductory through advanced biochemistry courses [2–5] and have involved experiments that include exploration of the Beer-Lambert * This work was supported by National Science Foundation Grants ILI 97-51426 and DUE-9850847, The Camille and Henry Dreyfus Foundation Special Grant Program in the Chemical Sciences, and University of Detroit Mercy alumni, allowing for the purchase of the laser-based scanning densitometer and the luminescence spectrometer. Preliminary accounts of this work were presented at the national meetings of the Society for Neuroscience and the American Society for Biochemistry and Molecular Biology, October 23–29, 1999 in Miami Beach, FL and June 4 – 8, 2000 in Boston, MA, respectively. ‡ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of Detroit Mercy, P. O. Box 19900, Detroit, MI 48219-0900. E-mail: casperml@udmercy. edu. 1 Problem-based learning initiative (PBLI) at Southern Illinois University School of Medicine at www.pbli.org/pbl/pbl_ essentials.htm. This paper is available on line at http://www.bambed.org
law [2], protein purification [3, 6], mapping uncharacterized mutations of the worm Caenorhabditis elegans [4], and studying various properties of a single enzyme [5]. These mini-projects occupy from one laboratory session [2] to the entire semester [3–5]. In the mid-1990s, the faculty of the Department of Chemistry and Biochemistry at the University of Detroit Mercy implemented project-based laboratories across the curriculum with the goal of incorporating one or more open-ended assignments of increasing difficulty into each laboratory course. In this way, even students who do not elect to undertake independent research are exposed to a wide variety of protocols and instrumentation in a professional, research-like environment. In their junior or senior year, biochemistry majors are required to take a twocredit, one-semester biochemistry laboratory course, which is concurrent with or subsequent to the second semester of the biochemistry lecture course. Because many of the students who enroll in the biochemistry laboratory course also need to master basic techniques, 5 years ago this course was redesigned to combine several structured experiments with two open-ended projects. All of the students who enroll in the biochemistry laboratory course have taken 1 year of general and organic chemistry, 1 year of general biology, and several advanced biology courses with laboratories. Many students also take the quantitative analysis lecture course concurrently with the biochemistry laboratory. Although the experiments in the laboratory are not coordinated with the biochemistry lecture course per se, the theory behind enzyme kinetics,
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BAMBED, Vol. 31, No. 5, pp. 303–307, 2003 TABLE I A typical solution-making and dilution problem
Suppose you need to set up an enzyme assay that requires adding a substrate, enzyme, and inhibitor according to the following chart. Assume the following. ● The final volume for all tubes is 200 microliters. ● The enzyme solution in buffer is available, and 20 microliters is added to all tubes. ● You may add additional buffer to tubes to bring them up to final volume. ● The limit of accuracy for the balance that you have is 10 mg. ● The limit of the pipettor available is 10 microliters. ● The molecular mass of the substrate is 250 g/mol, and that of the inhibitor is 200 g/mol. There is only 1 g of substrate and inhibitor left in the bottles. Tube no.
Final concentration of substrate
Final concentration of inhibitor
mM
nM
0 1 1 1 1 1 1
0 0 1 10 50 100 1 ⫻ 106
1–2 3–4 5–6 7–8 9–10 11–12 13–14
For each set of tubes below, explain EXACTLY what concentrations of stock solutions of substrate and inhibitor you would make to insure the proper final concentrations, and explain how you would make these solutions. Show your calculations. Also fill in the following table. Tube no.
[Stock substrate]
Substrate volume (microliters)
[Stock inhibitor]
Inhibitor volume (microliters)
Buffer volume (microliters)
Enzyme volume (microliters)
1–2
20
3–4
20
5–6
20
7–8
20
9–10
20
11–12
20
13–14
20
electrophoresis, and molecular exclusion and ion-exchange chromatography are covered in the first semester of the lecture course. Because most of the students in the laboratory course are taking it concurrently with the second semester of the lecture course, the second project, which deals with metabolic pathways, is late in the semester after metabolism has been completed in the lecture course. At present, the text Lehninger Principles of Biochemistry [7] is used in the lecture course. COURSE DESIGN
The biochemistry laboratory course has enrollments per semester of 9 –18 students, who are divided into teams of two to three students each. The team members are chosen either through random drawing or, when several of the students have already participated in an independent research project, through a process designed to obtain heterogeneous groups. The students work within the same team except for the first exercise, in which they work independently. In general, a short lecture precedes each new experiment where the experimental goals and general principles behind the procedures are explained. Quizzes for each experiment, except for the test/control project, are given 1 week after the completion of the experiment. The students complete a work sheet for the first exercise and present a poster for the final test/control project. For the other experiments, individual reports are required that are written in the style required by The Journal of Biological Chemistry. Final grades are based on the quality of the laboratory reports (75%), quiz scores (16%), peer grading (3%), notebook quality (3%), and performance in the laboratory (3%). The latter evaluates the student’s behavior in the laboratory such as arriving on time and active involvement within the group. The course meets for two 3-hour periods per week. The exper-
iments and the number of weeks allowed for each experiment are as follows. • Computer-based statistics and review of solution making (1 week). The rationale for the statistics and solution exercise is that students rarely have experience in the use of computerbased software such as Microsoft Excel for statistics. The class meets in the computer lab where the students use Microsoft Excel to perform linear regression analysis as well as to determine the variance and the significant difference between two sets of numbers. For all experiments, the students make all of their own solutions. The only solutions that are provided are those that use expensive reagents such as an enzyme where student errors would be too costly. Therefore, an exercise in calculating amounts of reagents to make solutions and dilutions (Table I) is included in the first laboratory. • Spectroscopy (1 week). The spectroscopy experiment allows students to compare the sensitivity of several procedures for protein concentration determination. The Biuret [8] and Lowry [9] methods illustrate that different assays have different effective protein concentration ranges. Other methods such as the Bradford [10] or bicinchoninic acid [11] assays could also be used. In addition, students explore the difference in sensitivity between spectrophotometric and spectrofluorometric methods by comparing the absorbance and fluorescence of 7-hydroxy-4-methylcoumarin using a Hewlett Packard diode array spectrometer (model 8452A) and a PerkinElmer Life Sciences luminescence spectrometer (model LS50B). The effect of pH on the fluorescence of 7-hydroxy-4-methylcoumarin is also studied.2 2 Molecular Probes, Inc. at www.probes.com/handbook/ sections/1001.html.
305 TABLE II Student evaluation of the biochemistry laboratory from 1999 –2002 The following questions will help us evaluate the effectiveness of the project-based format of the biochemistry laboratory so that we can modify its format for future classes. Your thoughtful answers to the questions are appreciated. Please answer the following questions by circling the number that is most appropriate. 5 ⫽ strongly agree
4 ⫽ agree
3 ⫽ neutral
2 ⫽ disagree
1 ⫽ strongly disagree RESULTS (n ⴝ 44) (Mean ⴞ S.D.)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12.
At the beginning of the semester, I liked the format of the lab. At the end of the semester, I liked the format of the lab. I liked the projects better than the structured labs. I would prefer more structured labs. I learned more from the projects than from the structured labs. Working with the group was better than doing it all myself. I prefer a three-person team. I like the way the teams were determined. Knowing that my group members were going to grade me on my participation was an incentive for me to work harder. I liked the point distribution for the labs and quizzes. Please give any other comments on the lab course. “Too much of the grade depends on the reports. I’d prefer more quizzes and statistical worksheets.” “The quizzes didn’t help much in learning.” (Note: numerous comments) “The 2 projects were a great chance to do some literature searches and to come up with a protocol—this is very applicable to a future career in research.” “Forced students to think independently, which increased the learning experience.” “This course, although it required a lot of time and energy, was extremely fun and academically rewarding. The best science class I’ve had here.” “This course was work, frustration, learning, experimenting, waiting, and basically one of my favorite classes.” “The projects were helpful, but frustrating, in the sense that we had to find the procedures ourselves. The structured labs gave us an idea of how to use techniques and what they measured, whereas the projects allowed us to work together to use the techniques. . .” Please give any other suggestions for improving the course in the future. “For the projects, perhaps find an article for the students to start with. Finding the first article was the hardest part of making a protocol.” “The first lab, I felt, was unnecessary.” “I think things would be greatly improved if more time was spent on statistical analysis.” “The lab report should be a group effort because it was worked on as a group.”
• Immobilized lactase (1 week). Students are exposed to the principles behind immobilized enzyme technology by setting up a small enzyme reactor for the hydrolysis of lactose [13]. • Enzyme kinetics (2 weeks). Students learn how to optimize reaction conditions (time, pH, temperature, enzyme and substrate concentrations) for an enzyme assay. They then determine the Michaelis constant (Km) and maximum velocity (Vmax) for the enzyme as well as the type and inhibition constant (Ki) for an inhibitor of the enzyme. The enzyme differs each year. To date, alkaline phosphatase, acid phosphatase, lysozyme, and -amylase have been studied. A commercial preparation of the enzyme is provided to the students. • Electrophoresis (1 week). SDS polyacrylamide gel electrophoresis is used to estimate the molecular weight of an unknown protein. The students use molecular weight standards to correlate molecular weight with relative mobility (Rf) and scan their gels on an Amersham Biosciences laserbased scanning densitometer (model PDS1) to determine the Rf values. This information is used to determine the molecular weight of an unknown protein. • Enzyme purification project (2 weeks). The same enzyme that was chosen in the enzyme kinetics experiment is used for this project. Several weeks before the start of the project, the student teams are asked to search the literature and then propose a three-step purification scheme. One of the steps must include a column chromatography procedure. The teams meet with the professors to present their purification schemes and give details as to the quantities of tissue, buffers, and chromatography resins so that sufficient materials can be ordered if necessary.
3.7 ⴞ 0.7 4.0 ⴞ 0.9 3.9 ⴞ 0.9 2.8 ⴞ 0.7 3.8 ⴞ 0.8 4.1 ⴞ 1.2 4.0 ⴞ 1.2 3.1 ⴞ 1.3 3.4 ⴞ 1.2 3.6 ⴞ 0.9
• Test/control project (6 weeks). Each year the professors choose the test and control tissues to be used. To date, tissue from normal/diabetic rats, normal/obese rats, male/ female rats, and rats/lake perch have been used. Several weeks before the project is to begin, students are told the type of tissue that will be available and are asked to propose a parameter that might be different between the two tissue groups and how they would test their hypothesis. The student teams are expected to do a literature search and present an outline of their proposal to the professors. Once their protocol is approved, they perform the experiments in at least triplicate so that they can use Student’s t test to determine whether a significant difference exists in the parameter between the test and control groups. Their results are presented at a departmental poster session where each team presents its poster and where students from other laboratory classes also present their work. RESULTS AND CONCLUSIONS
Since modifying the biochemistry laboratory course in 1999, 44 students (majors: 42 biochemistry, one chemistry, one biology) have successfully completed the course. Because of curricular demands, this laboratory is taught over only 1 semester, and at present, it is not possible to split the course over two semesters. However, because both structured experiments and open-ended projects are included, assessment of student preferences is possible. Students were asked to fill out an evaluation sheet at the end of the semester (Table II). From this evaluation, it was
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BAMBED, Vol. 31, No. 5, pp. 303–307, 2003
FIG. 1. Grades earned by 47 students who enrolled in the biochemistry laboratory course between 1999 and 2002.
apparent that students liked the laboratory format (Questions 1 and 2), but they preferred the projects to the structured laboratories and learned more from them (Questions 3–5). In fact, based on the agreement with Questions 3 and 4, only six students wanted more structured laboratories. Most students preferred working on a team as opposed to doing the work themselves (Questions 6 and 7), and although the students would have preferred to pick their own partners (Question 8), only one group over the 4 years had serious difficulties working together. Peer grading was not an important factor in student performance (Question 9), but these students were, for the most part, self-motivated individuals. Most students worked diligently, and their efforts were reflected in their final grades (Fig. 1). The abilities of these students allowed them to successfully compete for admission into professional/graduate schools and for professional employment (Fig. 2). From student comments, it is apparent that the quizzes were not liked. However, quizzes will continue to be given because it is a method to determine individual comprehension of the theory and techniques regarding the experiments. In addition, some students wanted group reports for the experiments. However, individual reports are another way of assessing individual understanding of the theory and results of the experiments. In addition, writing skills are such an important requirement for a successful career that individual reports will continue to be required. The poster session at the end of the semester began 2 years ago. A formal schedule was devised so that some teams were stationed at their posters while other teams and the professors visited them to ask questions and discuss results. In 2002, a joint session combining the biochemistry and second semester organic chemistry student posters was held, and the entire department was invited to attend. Some students did not feel that the first experiment dealing with statistics and solutions was worthwhile. However, none of the students were aware that Microsoft Excel could be used for statistics, and even those students who had used other statistical software needed the review. In addition, although students could define molarity, few of them appreciated that very dilute biological solutions often cannot be made directly because of the limits of the analytical balances. Serial dilutions (Table I) also caused problems for a substantial number of students.
FIG. 2. Career outcomes for 44 students who successfully completed the biochemistry laboratory course. Science employ indicates students who were hired with the B. S. degree into a science-related company. Non-sci employ indicates students with the B. S. degree who were professionally employed with a company that is not scientific.
The two open-ended projects differ in their degree of variability. With the enzyme purification project, student teams frequently accessed the same literature and so proposed identical purification schemes. For example, the year that acid phosphatase was partially purified, the six student teams all used an ammonium sulfate precipitation step, and five of the groups used a pH precipitation. Consequently, the professors insisted that each group use a different resin for the column chromatography step. As a result, different ion exchange and molecular exclusion resins were used by different teams. The test/control project offered more variability because each group had to choose a different parameter to study. In the year that male and female rat tissue was provided, four different enzymes were assayed as well as liver cholesterol levels (Table III). Even though two groups studied acid phosphatase, one group measured the specific activity of the kidney enzyme, and the other determined the sensitivity of the liver enzyme to tartrate. As students search for a parameter to study, several points have become apparent over the past 4 years. Some student teams did not want to think about characteristics and metabolism that might be different between the test and control tissue before beginning their literature search. Instead, they began an Internet search, often with disappointing results, or they searched a literature database and proposed an esoteric project that either required very expensive reagents or could not be completed within the 6-week time frame. On the other hand, student teams that discussed the differences between the test and control groups before the literature search had better success. For example, with the rat versus fish liver project, one group rationalized that the fish (which had been caught in the winter) might have less food available and therefore different levels of liver glycogen than the rat. Therefore, they limited their literature search to methods for glycogen determination. Their pro-
307 TABLE III Projects selected by students using male and female rat tissue Group
Parameter
1
Specific activity of lactate dehydrogenase (LDH) partially purified by differential centrifugation. LDH activity assayed by continuous monitoring of NADH conversion to NAD at 340 nm. Specific activity of alkaline phosphatase partially purified by n-butanol extraction. Alkaline phosphatase activity assayed by fixed time measurement of p-nitrophenol production. Specific activity of acid phosphatase partially purified by centrifugation. Enzyme activity determined by fixed time measurement of 7-methyl-4-hydroxycoumarin formation from 4-methylumbelliferone phosphate using the luminescence spectrometer. Tartrate inhibition of acid phosphatase partially purified by ammonium sulfate fractionation. Specific activity of glucose-6-phosphatase partially purified by centrifugation. Glucose-6phosphatase assayed by fixed time, colorimetric measurement of phosphate released from glucose-6-phosphate. Cholesterol was partially purified by acetone extraction. The cholesterol content was determined by a colorimetric assay using FeCl3.
2 3 4 5 6
Tissue
Statistically significanta
liver
yes
kidney
yes
kidney
no
liver liver
no no
liver
yes
a Results of student experiments using Student’s t test. Experiments were repeated a minimum of three times with tissue from different animals.
ject was approved, and they began the assays before some other groups had decided on a project. Another characteristic of the student teams is that many were reluctant to use the library and wanted to rely heavily on the Internet. Often their procedures were obtained from non-refereed sites, with unworkable results. In addition, the first draft of many of the projects lacked important experimental details such as quantities of tissue, buffer, or substrates. Also, the students frequently had to modify the literature procedure to accommodate the quantities of materials available in the laboratory. The professors made available a number of procedure manuals that would likely be found in research laboratories (for example, see Refs. 12 and 14) so students could obtain basic biochemical methods. Therefore, deadlines for the protocols were at least a week in advance of the start of the laboratory sessions. As others have noted [3], students who are exposed to traditional, cookbook experiments become accustomed to successful results and lack troubleshooting skills. Furthermore, rarely do students have the opportunity to design experiments to solve an experimental problem [3]. In a recent forum, industrial chemists listed problem-solving, team, and communication skills as among those most desirable in an employee [1]. The two projects described here allow students to hone these important skills, which are essential to success in a scientific career. This advanced biochemistry laboratory course was traditionally a senior level course. However, now juniors are highly encouraged to take it, and only those students whose schedules have serious conflicts are allowed to postpone it to senior year. The rationale is that many of our students opt to undertake an independent research project in the summer between their junior and senior years either with one of our faculty or at another university or in industry. This course prepares students well for such an undertaking, and the students have been successful in obtaining such summer research experiences.
Acknowledgments—We thank colleagues at the University of Detroit Mercy and Wayne State University for sharing rat tissue from animals sacrificed for other projects. We thank the undergraduate students who have participated in this course. REFERENCES [1] J. G. Otten, S. DeGrood, J. Landis, L. Wise, K. L. Perry, in M. A. Benvenuto, M. J. Mio, Eds. (2002) Proceedings of the 9 November, 2002 Meeting of the Michigan College Chemistry Teachers Association, pp. 9 –22, Detroit, Michigan. [2] D. B. Fialho, J. B. T. Rocha, C. F. Mello (1999) An introductory student-centred class to teach concepts related to spectrophotometry in a biochemistry practical laboratory course, Biochem. Educ. 27, 217–220. [3] L. M. Roberts (2001) Developing experimental design and troubleshooting skills in an advanced biochemistry lab, Biochem. Mol. Biol. Educ. 29, 10 –15. [4] J. Lewis (1999) The use of mini-projects in preparing students for independent open-ended investigative labwork, Biochem. Educ. 27, 137–144. [5] J. A. Smiley (2002) The most proficient enzyme as the central theme in an integrated, research-based biochemistry laboratory course, Biochem. Mol. Biol. Educ. 30, 45–50. [6] J. P. Loke, D. Hancock, J. M. Johnston, J. Dimauro, G. S. Denyer (2001) Teaching experimental design using an exercise in protein fractionation, J. Chem. Educ. 78, 1528 –1532. [7] D. L. Nelson, M. M. Cox (2000) Lehninger Principles of Biochemistry, 3rd Ed., Worth, New York. [8] A. G. Gornall, C. J. Bardawill, D. Maxima (1949) Determination of serum proteins by means of the biuret reaction, Biol. Chem. 177, 751–766. [9] O. H. Lowry, N. J. Rosebrough, A. L. Farr, R. J. Randall (1951) Protein measurement with the folin phenol reagent, J. Biol. Chem. 193, 265–275. [10] M. M. Bradford (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of ligand-dye binding, Anal. Biochem. 72, 248 –254. [11] P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, D. C. Klenk (1985) Measurement of protein using bicinchoninic acid, Anal. Biochem. 150, 76 – 85. [12] D. M. Bollag, M. D. Rozycki, S. J. Edelstein (1996) Protein Methods, 2nd Ed., Wiley-Liss, New York. [13] M. J. Allison, C. L. Bering (1998) Immobilized lactase in the biochemistry laboratory, J. Chem. Educ. 75, 1278 –1280. [14] J. M. Walker (ed) (2002) The Protein Protocols Handbook, 2nd Ed., Humana Press, Totowa, NJ.
