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Keywords:

  • Undergraduate;
  • protein purification;
  • research experience;
  • Taq polymerase

Abstract

  1. Top of page
  2. Abstract
  3. LABORATORY SERIES DEVELOPMENT
  4. STUDENT ASSIGNMENTS
  5. STUDENT PROFILE
  6. LEARNING OUTCOMES
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

We have developed a 9-week undergraduate laboratory series focused on the purification and characterization of Thermus aquaticus DNA polymerase (Taq). Our aim was to provide undergraduate biochemistry students with a full-semester continuing project simulating a research-like experience, while having each week's procedure focus on a single learning goal. The laboratory series has been taught for the past 7 years, and survey-based assessment of the effectiveness of the laboratory series was completed during the 2006 and 2007 fall semesters. Statistical analysis of the survey results demonstrate that the laboratory series is very effective in teaching students the theory and practice of protein purification and analysis while also demonstrating positive results in more broad areas of scientific skill and knowledge. Amongst the findings, the largest reported increases in knowledge were related to students' understanding of how patent law relates to laboratory science, a topic of great importance to modern researchers that is readily discussed in relation to Taq polymerase. Overall, this laboratory series proves to be a very effective component in the curricula of undergraduate biology and chemistry majors and may be an appropriate laboratory experience for undergraduates.

As more undergraduate students majoring in the natural sciences are interested in conducting research during their time in college, it is becoming ever more difficult to provide enough spaces in existing faculty research laboratories, especially at primary undergraduate institutions. In addition, with an increased interest in revising teaching methods to better prepare the next generation of laboratory researchers [1], the traditional biochemistry undergraduate laboratory experience's emphasis on particular techniques or concepts (i.e. pH, buffers, and amino acid titration curves), while fulfilling certain objectives, is often lacking in the important element of continuity as the semester progresses. Alternative approaches have involved a project design consisting of a series of connected laboratory experiments [2, 3]. Such approaches are advantageous in that they more closely typify the continuity between multiple steps and analytical skills encountered in the modern research laboratory and leave students with a more realistic viewpoint of the research process [4]. We have developed a 9-week biochemistry laboratory series for undergraduates that addresses these needs and incorporates the following Learning Objectives: 1) to develop skills in implementing techniques involved in protein purification and characterization and to gain an understanding of the principles of such techniques, 2) to develop organizational skills by advance preparation using flow chart design and detailed record-keeping by use of a laboratory notebook, 3) to foster analytical skills through data analysis and interpretation, 4) to strengthen verbal and written communication skills appropriate to scientific disciplines, and 5) to assist students in integrating their laboratory experience with relevant issues that face the scientific community, including academic, government, and industrial research and development. It is anticipated that these objectives will enable students to gain an experience of a research environment while also teaching them important skills and abilities that will further their overall training in the basics of a biochemistry laboratory. This laboratory series has been taught at the College of the Holy Cross for the past 7 years, and survey-based assessment of the effectiveness of the laboratory series was completed during the 2006 and 2007 fall semesters.

LABORATORY SERIES DEVELOPMENT

  1. Top of page
  2. Abstract
  3. LABORATORY SERIES DEVELOPMENT
  4. STUDENT ASSIGNMENTS
  5. STUDENT PROFILE
  6. LEARNING OUTCOMES
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

We chose to focus this entire laboratory series on the step-wise purification and characterization of a single enzyme. Thermus aquaticus DNA polymerase (Taq) was selected because it possesses several advantageous properties. First, Taq is a thermostable enzyme that enables chromatographic purification at ambient temperature thus eliminating the cumbersome situation of access to a cold room or chromatography cabinet space by multiple students. In addition, because of this great stability, Taq is quite unlikely to lose all of its activity during the rigors of purification. Second, bacterial over-expression systems have made Taq polymerase an easy enzyme to purify. While successful single-step methods of purification have been published [5–7], we have adapted a traditional, step-wise purification strategy originally developed by Engelke et al. [8] for this laboratory series. Our rationale was to provide straightforward procedural learning objectives so that students could focus their efforts on a single objective each week with the combination of each week's objectives leading to the final analyzed protein sample. The anticipated outcome upon completion of the series would be that students would have developed a better understanding of the purpose of each technique in the overall purification and analysis scheme.

The overall laboratory series consists of the following general week-to-week progression, with each week's procedure feeding in to the next week (see also flowchart in Fig. 1).

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Figure 1. Flowchart depicting the major experimental procedures conducted during each laboratory period of the purification and analysis series. As much as possible, each week has a primary experimental focus that allows students to explore a particular type of technique without becoming confused by also learning other methods during the same laboratory period. Although students must successfully complete all of the purification procedures (shaded red) from weeks 1 to 4 to produce a sample of purified Taq protein that can be subjected to the analysis labs (shaded green) in weeks 5–9, the analysis laboratory procedures have been written in such a way that they include positive and negative control samples that allow all students to complete all 9 weeks of laboratory and still learn all of the experimental methods related to the purification and analysis.

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Each week is intended to contribute to the development of skills and concepts for mastering a specific technique (Learning Objective 1: Laboratory techniques and skills). Week 1—preparation of starting material, week 2—purification by precipitation, week 3—purification by chromatography, week 4—ultrafiltration and dialysis, week 5—protein determination, week 6—polyacrylamide gel electrophoresis, week 7—western blotting, weeks 8 and 9— polymerase chain reaction (PCR)-based enzyme assay and wrap-up. Because of the extent of procedures required for this 9-week series, detailed methods have not been included in the text of this paper. However, detailed procedures and preparatory notes for the entire laboratory series will be freely shared by the corresponding author upon request.

Weeks 5–9 are particularly effective in providing students with the opportunity to collect, analyze, and interpret data (Learning Objective 3: Data analysis and interpretation). This will enable them to assess whether Taq polymerase was purified and whether it retained enzymatic activity. The exercises performed in week 5 allow the students to assess a number of different protein determination methods, each of which is based upon the principle of the Beer-Lambert law. The four quantitative spectroscopy methods employed are direct quantification by measuring A280, the bicinchoninic acid assay, Bradford assay, and a Bio-Rad DC (Bio-Rad, Hercules, CA) (modified Lowry) assay. To determine the identity and purity of the protein sample, the students separate their samples by SDS–PAGE electrophoresis in week 6. The gel is loaded in duplicate, along with a commercially available sample of Taq protein, which allows them to perform a Coomassie stain of one set of samples to determine purity of the sample and transfer the second half of the gel to a nitrocellulose membrane for Western blot analysis. In week 7, the nitrocellulose membrane is probed for Taq using an anti-Taq antibody and an HRP-conjugated secondary (Fig. 2, Panel a). Based on the Coomassie stain and the Western blot signal, the students can assess the purity of the sample as well as determine if Taq protein is indeed present in the sample. The series concludes with a functional assay of the purified Taq sample. A dilution series of the purified sample is prepared and the relative activity of the enzyme in the sample is compared against a commercially available Taq enzyme in a PCR amplification assay (Fig. 2, Panel b).

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Figure 2. Examples of data generated by student groups completing the Taq polymerase purification and analysis laboratory series. Panel a depicts a Western blot conducted by a student group to analyze their sample of purified Taq polymerase. Concentrations were determined by Bradford assay. Lanes 1–3 show a series of purified Taq polymerase, Lane 4 is Taq polymerase (2 μL) purchased from Promega (Madison, WI). After transfer from the SDS–PAGE gel, the Western blot was probed with TaqStart antibody (Clontech, Mountain View, CA) followed by incubation with goat anti-mouse-HRP conjugate and ECL detection (GE Healthcare, Piscataway, NJ) to visualize the Taq on the blot. Approximate molecular weights are illustrated. Note that both the commercial and student-purified Taq are ∼94 kDa, as would be expected for full-length Taq polymerase. Panel b depicts an ethidium bromide stained 1.5% agarose gel run by a student group on DNA samples generated from a PCR reaction using their purified preparation of Taq in a dilution series (Lanes 2–6) compared to a commercial sample of Promega Taq polymerase (Lane 7). TrackIt 1kb ladder (Invitrogen, Carlsbad, CA) is shown in Lane 1 and a no template control (Lane 8, NTC) was included as a negative control. The template and primers used for the reaction were designed to produce a 120 bp product, which can clearly be seen in Lanes 2–4 and Lane 7.

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It should be noted that the series, in its current form, lacks a true quantitative enzyme assay that would allow students to directly calculate enzymatic activity, as well as kinetic constants. The enzymatic activity of commercial preparations of Taq polymerase, like most DNA polymerases, are typically characterized by determining the quantity of radiolabeled-deoxyribonucleotide incorporated into acid precipitable material by a known quantity of enzyme over a defined period of time [9]. While the authors agree that the addition of an enzyme assay would be a very useful addition to the overall series, our initial attempts to adapt a quantitative, nonradioactive enzyme assay for Taq polymerase [10] to the series have not been successful. New attempts to develop these procedures are currently being conducted at the University of Connecticut, where the laboratory course will also be taught during the fall semester of 2009, and we are hopeful that this component can be added in the future to compliment the existing PCR-based assay run during weeks 8 and 9.

STUDENT ASSIGNMENTS

  1. Top of page
  2. Abstract
  3. LABORATORY SERIES DEVELOPMENT
  4. STUDENT ASSIGNMENTS
  5. STUDENT PROFILE
  6. LEARNING OUTCOMES
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

The central focus of the student assignments is to foster integration of the present laboratory experience with the research literature (Learning Objective 5: Integrate laboratory experience with relevant issues that face the scientific community). The assignments range in format from keeping a laboratory notebook to an investigation into current controversies surrounding intellectual property. To encourage students to develop good laboratory practice procedures including mapping out experiments in advance and organizing collected data effectively (Learning Objective 2: Develop organizational skills and detailed record-keeping by use of a laboratory notebook) laboratory notebooks are evaluated both early in the semester and at the end. Initially, students are given detailed formative comments with summative comments reserved for the end of the series. In addition, students follow up each week's laboratory with responses to questions designed to provoke their thinking about the theory, advantages, and disadvantages of the particular technique employed that week.

To encourage engagement with the primary literature as well as the ability to present scientific material orally (Learning Objective 4: Strengthen verbal and written communication skills), students are required to initiate a search of the primary literature to compare and contrast the different approaches and rationale for protein purification, and present a 5–10 minute synopsis in class based upon the paper they selected. These brief synopses are informative for the students and maximize effective use of the “open” time during incubation steps or centrifuge runs.

To encourage students to develop writing skills in the discipline (Learning Objective 4), students are required to submit the compilation of their purification and characterization of Taq polymerase in manuscript format. Five weeks into the laboratory series the students are presented with the Instructions to Authors document based loosely on the Journal of Biological Chemistry requirements, as criteria for manuscript submission. Following discussions about the Instructions and implementing them, students gradually come to understand the process that scientists use in preparing research papers.

Lastly, to bridge the students' laboratory experience with concrete issues faced in the biotechnology industry, the students are asked to calculate the commercial value of their final preparation of Taq polymerase based on the results of their final PCR assay and the current marketing cost for Taq. This exercise promotes discussion of the various factors involved in the development, distribution, and marketing of commercial reagents and pharmaceuticals (Learning Objective 5). Moreover, the widespread use of the PCR in many biological disciplines enables undergraduate students to appreciate the practical significance of protein purification and characterization. In addition, the patent battles that have surrounded Taq polymerase provide an easy opportunity to engage students in a discussion of patent law. As the Taq patent battles have been covered extensively in major scientific journals, such as Science and Nature [11–17], students investigate and write a two-page summary of the controversies surrounding the patent challenges to the licensing of Taq. This assignment prompts students to become engaged in a lively discussion about patent law.

STUDENT PROFILE

  1. Top of page
  2. Abstract
  3. LABORATORY SERIES DEVELOPMENT
  4. STUDENT ASSIGNMENTS
  5. STUDENT PROFILE
  6. LEARNING OUTCOMES
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Students who elect to enroll in Biochemistry Laboratory concurrent with the lecture course are either juniors or seniors with a minimum of four semesters of chemistry with laboratory, including two semesters of organic chemistry. Most of these students thus possess basic laboratory skills acquired from these courses. To supplement these basic skills with several biochemical- orientated experiences before beginning the multi-week series, we precede the 9-week series with 2 weeks of nonlinked, “skill building” laboratories focused on the basics of using micropipets, pouring chromatography columns, conducting dialysis and the like. If the inclusion of these types of initial laboratories is not possible, then basic demonstrations by the instructor should be all that is necessary to build the level of competence required for this laboratory series.

LEARNING OUTCOMES

  1. Top of page
  2. Abstract
  3. LABORATORY SERIES DEVELOPMENT
  4. STUDENT ASSIGNMENTS
  5. STUDENT PROFILE
  6. LEARNING OUTCOMES
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

To formally assess the effectiveness of this laboratory series in teaching students both the theory and practice of protein purification and analysis, a pre- and postsemester survey was conducted with the students taking the laboratory course during the fall semesters of 2006 and 2007 (12 students each year). The survey consisted of a series of 25 self-assessment questions that asked students to rank their confidence in their ability to explain or conduct biochemistry and/or research related activities (questions 1–14) and to rank their individual rating of skill in using specific pieces of equipment or in conducting specific laboratory procedures (questions 15–25). Each question allowed students to select a numerical rating from zero (low confidence or skill) to five (high confidence or skill). Students voluntarily filled out the survey both before and after completing the laboratory series. To ensure that responses remained anonymous, students were asked to provide and write a two-word phrase on the back of the presurvey. For the postsurvey, students were then asked to circle their phrase from among a list of choices to permit direct correlation of pre- and postsurveys of the laboratory series. The assessment survey forms and overall assessment strategy were reviewed by the College of the Holy Cross Human Subject Research committee and found to fall under exempt status. The survey forms are available by request from the corresponding author of this paper.

Paired pre- and postsurvey responses were analyzed using the Wilcoxon nonparametric signed-rank test. In considering the 2006 data, all average (post–pre) values were positive numbers, and one-tailed p values from the Wilcoxon test indicated that the overall change from pre- to postsurvey response was significant (p ≤ 0.05) for all questions with the exception of two (Table I). These two questions,

Table I. Results of student pre- and postsurveys taken during the fifth and sixth years that the taq laboratory series was taught
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“Work with other students in a laboratory group” and “Use a spectrophotometer and a plate reader”, had an average presurvey responses of 4.50 and 3.75, respectively, indicating that the students entered this laboratory series already having fairly high confidence in these areas. The lack of a significant increase was therefore not surprising.

The 2007 assessment data is, for the most part, in agreement of the findings from the 2006 data set. Again, all average (post–pre) values were positive numbers. TheWilcoxon test indicated that the overall change from pre- to postsurvey response was significant (p ≤ 0.05) for all questions with the exception of three (Table I). In 2007, there were no significant differences in the pre- and postsurvey results for the questions “Work with other students in a laboratory group”, “Take exams and write papers in science classes,” and “Use an FPLC.” As in 2006, the likely reason for the lack of significant change on the first two questions relates to the average presurvey responses being high, 4.42, and 4.08, respectively. The third question, “Use an FPLC” is not significant because very few students reported confidence in this procedure in the postsurvey (average of 1.50), accurately describing the fact that an FPLC demo was not included in the 2007 fall semester due to limitations of time.

Based on the self-reporting of students on the pre- and postsurveys during both the 2006 and 2007 fall semesters, this laboratory series is effective in several important areas (see Table I), all of which are important aspects of research involving biochemical techniques such as chromatography, SDS–PAGE and Western blot analysis. Notably, significant increases were also reported in written scientific communication skills as well. Students reported an increased confidence in reading and analyzing the primary scientific literature, including the ability to explain the theory and implementation of biochemical techniques, keeping a laboratory notebook, and preparation of their data as a scientific manuscript for publication. Students also reported increased confidence in many cognitive skills such as experimental design, troubleshooting problems, and data analysis. Finally, the survey question that showed the largest increased pre- to postsurvey increase was “Describe how patent law relates to laboratory science”. Fourteen of the 24 students rated their confidence on this item in the presurvey as “0”, but felt confident enough to self-report a score of “3,” “4,” or “5” on the postsurvey. Thus, the discussion of patent law provides an additional benefit to this laboratory series curriculum as it introduces students to an important issue facing the biotechnology industry.

Students enrolled in this laboratory series have the opportunity to experience many aspects of an independent research laboratory. This both teaches students practical research skills and provides them with an experience that may enable them to decide whether biomedical-related research would be a potential career option that they would enjoy and find personally fulfilling.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. LABORATORY SERIES DEVELOPMENT
  4. STUDENT ASSIGNMENTS
  5. STUDENT PROFILE
  6. LEARNING OUTCOMES
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Overall, the 9-week Taq polymerase purification and analysis laboratory series described in this paper represents a successful approach to the laboratory training of undergraduates. The series increased their confidence in many skill areas that are key to being a successful researcher in molecular and cell biology and provided them with hands-on experience of biochemical techniques in a research-like setting. The laboratory series has become an integral component of the curricula in the Biology and Chemistry Departments at Holy Cross and we encourage its adoption by faculty at other undergraduate institutions. Detailed weekly procedures and laboratory preparatory notes are freely available by request from the corresponding author. In addition, instructors who are interested in adopting these procedures are encouraged to contact the authors with any questions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. LABORATORY SERIES DEVELOPMENT
  4. STUDENT ASSIGNMENTS
  5. STUDENT PROFILE
  6. LEARNING OUTCOMES
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

The authors thank Kelly Wolfe-Bellin and Robert Bertin for their help with statistical analysis. The authors thank the undergraduate teaching assistants who have participated in the preparation and instruction of the laboratory course, and the Holy Cross undergraduate students who have taken the Biochemistry I Laboratory course in the past 7 years for their enthusiasm and their feedback that helped to push forward the development of this laboratory series.

REFERENCES

  1. Top of page
  2. Abstract
  3. LABORATORY SERIES DEVELOPMENT
  4. STUDENT ASSIGNMENTS
  5. STUDENT PROFILE
  6. LEARNING OUTCOMES
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES