Investigating the Determinants of Substrate Binding through a

Article Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX

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Investigating the Determinants of Substrate Binding through a Semester-Long, Project-Oriented Biochemistry Laboratory Course Catherine A. Sarisky and Timothy W. Johann* Department of Chemistry, Roanoke College, 221 College Lane, Salem, Virginia 24153, United States

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S Supporting Information *

ABSTRACT: A semester-long hypothesis-driven laboratory project for second-semester biochemistry has been developed. Working independently, students propose a hypothesis about the role of one amino acid residue in the active site of 5,10methenyltetrahydrofolate synthetase (MTHFS). They then test this hypothesis by site-directed mutagenesis of a plasmid encoding the wild-type enzyme, overexpression and purification of the mutant protein, and detailed kinetic characterization. Student success in this laboratory course requires attention to detail and careful preservation of materials from week to week. Controls are carefully selected to allow diagnosis of experimental failures, and students are required to discuss them in their laboratory reports. The combination of independent work coupled with experiments in which results from one week influence the next provides students with a model of professional research activities that is absent from traditional laboratory experiments. KEYWORDS: Upper-Division Undergraduate, Biochemistry, Enzymes, Laboratory Instruction, Hands-On Learning/Manipulatives students first isolate and kinetically characterize ribose 5phosphate isomerase from spinach and then clone and overexpress the Escherichia coli version of the enzyme.2 Witherow reports a 10 week introductory biochemistry laboratory in which students overexpress mutant and wildtype alkaline phosphatase from existing plasmid constructs and then characterize the kinetics of the commercially purchased enzyme.3 Craig reports a 12 session laboratory series in which students overexpress and kinetically characterize threonine dehydrogenase from an existing plasmid, which is followed by 6 sessions devoted to student projects.4 Farnham and Dube report a first-semester biochemistry laboratory series focused on Helicobacter pylori urease in which students overexpress, partially purify, and characterize wild-type urease, before starting a three-session urease-related project.5 Taylor et al. describe an experimental series in which students examine a Gleevec-resistant tyrosine kinase mutant using a lactate dehydrogenase- and pyruvate kinase-coupled assay.6 This report describes a semester-long (12 sessions) project for a second-semester biochemistry course in which students determine which amino acids are important for the binding of ATP to the enzyme 5,10-methenyltetrahydrofolate synthetase (MTHFS). This laboratory course is novel in that students initially hypothesize which amino acid side chains are important on the basis of crystal-structure data, and then

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n traditional science laboratory courses, students perform each week’s experiment as a stand-alone task. In these experiments, products are discarded at the end of the laboratory period, resulting in minimal student concern for the success of the experiment. This process can leave students without an understanding that the product of one reaction is the substrate for the next. As each student or team in a traditional laboratory performs an identical experiment with little or no emphasis on obtaining a useful product, students may feel minimal intellectual ownership of the results. This contributes to a lack of internal motivation to make careful measurements and to pay close attention to detail. Lewis observed, and our experience is similar, that undergraduate students are unaccustomed to taking responsibility for collecting reliable data and have unrealistic expectations about how quickly results can be achieved.1 While work carried out by research scientists is hypothesis-driven, an emphasis on the formulation and evaluation of a hypothesis is often missing from traditional laboratory experiments. As such, traditional laboratory courses poorly model some of the practices that are essential in the work of professional researchers. One solution to this dilemma is to provide students with a laboratory course that contains one or more multiweek projects or one project that spans an entire semester. Multiple examples of biochemistry laboratory courses with these features have been previously described.2−13 Jewett and Sandwick describe a thematically connected series of experiments for a twice-weekly biochemistry laboratory in which © XXXX American Chemical Society and Division of Chemical Education, Inc.

Received: January 10, 2018 Revised: July 18, 2018

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DOI: 10.1021/acs.jchemed.8b00028 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

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Table 1. Assessment of Pedagogical Goals Assessment Goal (1) (2) (3) (4)

Use a series of techniques to test a hypothesis Use data including controls to troubleshoot laboratory failures Master complex solution making (calculations and techniques) Learn the theory and practice of common techniques

Report 1a 2, 4 9c, 10c 9 4, 5

Report 2

Report 3

3 14, 15

3 12

4−6, 8, 12

4−11

Report 4

Exams and Worksheets

6 7 4, 5 4, 5

Final examb,c Midsemester exams and worksheetsb Final exam,b,c ACS examc ACS exam,c midsemester examsb

a

Numbers reference the item in the report directions. See the Supporting Information, pp S45−S49, for prompts for all report items. bSample questions are included in the Supporting Information, p S59. cResults included in this report.

The MTHFS enzyme is relevant to human health and the treatment of disease, which provides an additional tool to motivate student engagement in the project. Tetrahydrofolates are cofactors in one-carbon metabolism. These molecules donate carbons of various oxidation states in the synthesis of nucleotides, amino acids, and metabolites essential to DNA repair.14,15 MTHFS catalyzes the conversion of 5-formyltetrahydrofolate (5-formylTHF or folinic acid) into 5,10methenyltetrahydrofolate. The substrate, 5-formylTHF, is produced by a side reaction of serine hydroxymethyltransferase and is thought to be a storage form of tetrahydrofolate. The reaction catalyzed by MTHFS adds 5-formylTHF back into the active tetrahydrofolate pool.16,17 MTHFS is clinically relevant in that 5-formylTHF, under the drug name leucovorin, is administered with other pharmaceuticals in the treatment of cancer and parasitic infections. It provides an alternate path for the production of active tetrahydrofolates when their synthesis from folic acid is inhibited.14,18

they use the next 11 sessions of the laboratory course to test their hypotheses using site-directed mutagenesis and protein overexpression and purification, followed by determination of the kinetics of their individual mutant proteins. This series is a linear chain of experiments with the results or products of each experiment immediately required for the next. Our pedagogical goals for the laboratory sequence are that students will (1) demonstrate an understanding of how multiple techniques can be serially applied to test a hypothesis; (2) use data, including positive and negative controls, to diagnose laboratory failures; (3) master complex solution-making in terms of both calculations and practice; and (4) become knowledgeable of the theory and skilled in the practice of a series of laboratory techniques common to molecular biology and biochemistry. As shown in Table 1, progress toward these goals is monitored throughout the semester on laboratory reports, worksheets, and exam questions in the associated lecture course. Although other semester-long projects in which students focus on a single enzyme have been published, the project reported here offers several distinct advantages. First, students propose and conduct their own site-directed mutagenesis, leading to the production and characterization of mutant proteins. Several of the other reported projects do not have students conduct their own mutagenesis or have them only study a well-characterized commercial or wild-type enzyme; thus, all students in the laboratory may be testing the same enzyme. Second, production of the product, 5,10-methenyltetrahydrofolate, is determined by direct UV−vis measurement, allowing easy determination of initial rates using 1 min data collection. Because the product’s absorbance maximum is 356 nm, it is possible to conduct kinetics trials with inexpensive, small UV−vis spectrophotometers (such as a Spectronic 20), which are available in numbers sufficient for an entire laboratory section in many institutions. Third, the experiments in this project require minimal instrumentation other than the spectrophotometers, which should make the experimental series possible to implement in a variety of contexts without major instrumentation acquisitions. (Although Roanoke College students use a Nanodrop at a few points, its use is not essential, and workarounds are offered in the Instructor’s Notes, Supporting Information.) As designed, these experiments require 12 laboratory meetings and so could serve as an entire second-semester laboratory course or a half-semester module at an institution with two laboratory meetings per week. In contrast to some previously reported project-based experiments5,7,8 in which each student or team performs different techniques to address a student-proposed question, students in this course all use the same set of techniques, which allows the instructor to offer a relevant prelaboratory lecture and to manage as many as 22 students working independently while still engaging students in testing their own hypotheses.

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CURRICULAR CONTEXT Roanoke College is a small, primarily residential liberal-arts college. Biochemistry courses are taught within the Department of Chemistry, which is certified by the American Chemical Society; the Biochemistry major is a collaboration between the Departments of Chemistry and Biology and is certified by the American Society for Biochemistry and Molecular Biology. This particular laboratory course is a required part of the second semester of biochemistry and runs concurrently with the lecture. The lecture is primarily focused on central metabolic pathways (carbohydrates, lipids, amino acids, and nucleic acids) as well as detailed discussions of genes, DNA and RNA metabolism, and gene expression. Laboratory topics are largely separate from the lecture except for a day or two of discussion on the roles of tetrahydrofolates in amino acid and nucleic acid metabolism. One laboratory section of the second semester of biochemistry is offered each year, with enrollment ranging from 10 to 22 students. The cohort is a mix of biology, biochemistry, chemistry, and psychology majors, with biology and biochemistry majors making up the majority of the students. The class is required for the biochemistry major, and most students enrolled have a healthcare career focus. Students work individually in the laboratory, a new experience for most of those who have not participated in independent research. Although some experiments require students to work outside the laboratory period (see the Supporting Information for the laboratory manual), none of them run substantially over 3 h of work per week. Most laboratory meetings begin with a 20−30 min lecture, in which special emphasis is placed on how that week’s work fits into the overall project. Student mastery of laboratory concepts is assessed primarily through four formal reports, with additional appraisal through questions on B



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worksheets and lecture exams, including the final exam. To encourage careful work, roughly 10% of each student’s laboratory grade is based on correct execution of the experiment, resulting in demonstrable product and expected results for the controls. Proper notebook technique is assessed through laboratory-notebook quizzes. Prelaboratory assignments are occasionally required, especially when students will be making their own solutions. Instructions for proper laboratory-notebook technique and for all four formal laboratory reports are provided in the Supporting Information along with a sample laboratory-notebook quiz. Although the laboratory component of the second semester of biochemistry makes up 20% of a student’s grade for the entire course, it comprises roughly 50% of the effort.

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Figure 1. Student-generated figure illustrating the following hypothesis: “The K3A mutant is important to ATP binding by MTHFS. The positive charge of the lysine is nearby to the negative charges of the oxygens on the phosphates on the ATP. Thus through charge charge interactions this binding would be stabilized. Based on this evidence, I would predict that by changing this lysine to alanine, MTHFS would bind ATP more weakly, and thus raise the Km.”

EXPERIMENT OVERVIEW As shown in Table 2, in the first week of the course, students are introduced to MTHFS with an emphasis on its relevance in Table 2. Overview of Experiments Week 1 2 3 4 5 6 7 8 9 10−12

Experiment Choose an amino acid, formulate a hypothesis, and design primers Change the codon for a chosen amino acid to the codon for alanine by site-directed mutagenesis Transformation of chemically competent bacteria with mutated plasmid DNA Isolation of mutated plasmid DNA from bacteria Analysis of DNA-sequencing data and transformation of bacteria with mutant DNA Induction of protein expression Protein extraction using the freeze−thaw technique Protein purification using immobilized-metal-affinity chromatography Protein purification using size-exclusion (gel-filtration) chromatography Kinetic analysis of the MTHFS mutant

required to come back to laboratory for 15 min to degrade the wild-type plasmid templates using the restriction enzyme DpnI. To confirm that the wild-type DNA has been mutated, students must generate and purify enough plasmid for DNA sequencing. The first step in this process occurs in week 3 when students transform chemically competent E. coli (XL1blue strain, made competent by the Inoue method21) with their mutated MTHFS plasmids. They also run agarose gels of the products from their site-directed-mutagenesis reactions. Although these gels do not have the resolution to verify that mutations were made, they can be used to determine if an appropriately sized plasmid has been isolated. The first formal report is due after this laboratory experiment, and the results from the agarose gels are useful for the interpretation of the transformation results. Students start cultures from their transformed bacteria the day before the fourth laboratory period, and they purify plasmid DNA from those cultures during the laboratory period using a commercial miniprep kit (Wizard Plus SV Minipreps DNA Purification System from Promega). The mass of DNA purified is quantitated using UV−vis spectroscopy, and students prepare samples to be shipped off campus for DNA sequencing. The instructor provides the results online before the next laboratory meeting. In the fifth laboratory meeting, students analyze the results from DNA sequencing to verify that they successfully produced a mutant MTHFS gene with their codon of interest changed to the codon for alanine. They then transform an E. coli strain (Rosetta 2 (DE3)) with their plasmid containing this mutant gene. The second formal report is due after this experiment. The next 2 weeks are devoted to growing bacteria, inducing them to produce a mutant protein, collecting cell pellets, and extracting the protein from the cells. Bacterial growth and induction represent one experimental block that takes much longer than 3 h and must occur over 3 consecutive days; however, much of this time is waiting, and the entire activity takes fewer than 3 h of active work for the students. Briefly, bacterial cultures in 100 mL of LB medium are grown at 37 °C to an optical density of 0.8−1.0 absorbance units at 600 nm; this is followed by the addition of isopropyl β- D -1-

the treatment of human disease. They are presented with the research question “Which amino acids of MTHFS are important to the binding of ATP?” Students use PyMOL to inspect the cocrystal structure of MTHFS,19,20 formulating hypotheses of how an amino acid side chain of their choosing helps to bind ATP in the active site of the enzyme, such as is shown in Figure 1. Students then design complementary DNA primers for changing the codon for that amino acid to the codon for alanine. The long-range goal is to produce a mutant protein that replaces their chosen amino acid with alanine to test their hypotheses about ATP binding. In the second week, students are provided with a plasmid containing the wild-type gene for MTHFS from M. pneumoniae, kindly provided by the authors of ref 19 and available from Addgene (pSKB3.MPN348). They then change the codons of their chosen amino acids to that for alanine through site-directed mutagenesis (an adaptation of the QuikChange protocol using Phusion polymerase, as described in the Supporting Information), using the primers designed in the previous week. Given the narrow focus of the research question, there are fewer than 10 relevant amino acids; the need to order new primers is obviated after the first few years as the appropriate primer stocks have already been obtained. As the site-directed-mutagenesis reactions take several hours, the instructor is responsible for placing completed reactions in the freezer. Before the next laboratory period, students are C



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Figure 2. Sample student data from the kinetic characterization of a student-selected mutant, K120A, showing a Km for ATP of 600 μM based on the Lineweaver−Burk plot or 970 μM from fitting with the Michaelis−Menten equation. The student result broadly agrees with previous work showing that K120A’s Km is 1200 μM.22 For comparison, the wild-type enzyme’s Km is 76 μM. Although the student result does not perfectly reproduce the literature value, both values are consistent with a substantial impairment in binding to ATP for this mutant.

thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Induction proceeds overnight at room temperature and is followed by centrifugation and freezing of the bacterial cells. The next experiment involves the lysis of bacteria using a freeze−thaw technique to extract mutant proteins, leading to 1 mL of lysate in phosphate-buffered saline. This process also takes much longer than 3 h, with most of that time devoted to waiting. The scheduled laboratory meetings for these two experiments are both canceled; the laboratory is left open during business hours, and students plan their time to best fit their individual schedules. For even greater flexibility, the 2 weeks for these experiments straddle Spring Break. Students can complete both experiments in 1 week or across 2 weeks as best works for them. In the eighth week, the class reconvenes in the formal laboratory period to purify their mutant enzymes from extracts using metal-affinity chromatography. The plasmid includes the codons for an N-terminal polyhistidine tag (MGSSHHHHHHDYDIPTTENLYFQGH) in frame with the MTHFS gene. This procedure is carried out in batch mode in 1.5 mL centrifuge tubes, using centrifugation to separate the TALON resin (with MTHFS bound) from the supernatant in lieu of column chromatography. Purified MTHFS is released to the supernatant using a high concentration of imidazole that must be removed in the next experiment. Size-exclusion chromatography is used to remove the imidazole from the mutant MTHFS sample in the ninth week. This is carried out using a 4.25 cm drip column. The concentrations of protein in the individual fractions are quantitated by UV−vis spectroscopy. Products from this experiment and those from previous weeks are run on denaturing polyacrylamide gels to evaluate the effectiveness of the purification. The third formal report is due after this experiment. The final 3 weeks of the laboratory course are devoted to kinetic analysis of the MTHFS mutants. Students are given only general directions and buffer conditions and must determine the Km for ATP and the kcat values of their mutants. This work includes complex solution preparation, determination of proper enzyme dilution, and discovery of the proper ATP-concentration range to use for the final data collection.

Student who demonstrate inactive mutants, in triplicate and with proper controls, are provided with samples of wild-type MTHFS to kinetically characterize. The final formal report is due after this experiment.

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HAZARDS

There are multiple hazards associated with this laboratory course. Students are required to wear goggles, gloves, and laboratory coats for all experiments. Long hair must also always be tied back. Physical dangers include Bunsen burners that are used to sterilize glass and inoculating loops at several points. Liquid nitrogen can cause cryogenic burns and frostbite. Many of the hazards in this course are associated with gel electrophoresis. Outside of the risk of electric shock, there are several dangerous chemicals. Sodium dodecyl sulfate (SDS), 2mercaptoethanol (βME), and acrylamide are toxic if swallowed or inhaled. Acrylamide is also toxic on skin contact, is a skin allergen and eye irritant, and is a suspected reproductive toxin and carcinogen. 2-Mercaptoethanol is toxic on skin contact, is a skin irritant and an allergen, and causes serious eye damage. SDS is a skin and respiratory irritant and causes serious eye damage. Gel Code Blue (used to stain polyacrylamide gels) is toxic upon ingestion and skin contact, is a skin irritant, and causes serious eye damage. SYBR Safe DNA gel stain is flammable and contains components that may be absorbed through the skin. In cell-culture work, kanamycin monosulfate, IPTG, and bleach are of note. Kanamycin monosulfate is a reproductive toxin. IPTG is a suspected carcinogen, and bleach is a skin irritant and causes serious eye damage. The DNA-isolation kit contains components that are allergens, reproductive toxins, and skin and respiratory irritants and components that are damaging to the eyes and toxic upon ingestion. Folinic acid and Triton X-100 are dangerous chemicals used in the kinetics experiments. Folinic acid is a skin, eye, and respiratory irritant as well as an allergen. Triton X-100 is a skin irritant, is toxic upon ingestion, and can cause serious eye damage. D



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question, with no student scoring lower than 73% of the points possible. Students have also been asked to explain the overall goal of the project on the final exam (see p S60). For 2017 and 2018 (n = 24), they averaged 92% of possible points, with 19/ 24 students scoring full credit on this question. Substantial emphasis is placed on these concepts during the semester. In addition to laboratory-report prompts, these concepts are reviewed at the beginning of each laboratory meeting. That students use available data (controls and other students’ results) to diagnose experimental failures is our second pedagogical goal for the course. The importance of controls is emphasized in the prelaboratory lecture and in the laboratory manual. The site-directed mutagenesis reaction in the second week of the laboratory involves the pipetting of multiple small volumes to make solutions. This particular point in the laboratory sequence has historically been rich in failure that is not discovered until the imaging of an agarose gel at the end of the third week of work. Thus, many students will have failed site-directed-mutagenesis reactions and will subsequently fail to transform bacteria. The directions for the first laboratory report (p S47) instruct students to address nine possible sources of error across these 2 weeks using data from controls and other students’ results. Reports from 2017 and 2018 were scored on a 5 point rubric (pp S60 and S61). Across 45 student laboratory reports, the average score for these items was 3.7, with some students earning one or more 0 scores for items not discussed in their error discussion. It is not clear whether these students chose not to attempt the analysis required because they did not understand it, or if they simply failed to carefully read the laboratory-report directions. When these nonattempts (40 of 348 scores) are excluded, the average score rises to 4.1. Regardless of the inclusion or exclusion of 0 scores, students are averaging near a value of 4.0 (corresponding to only minor errors), indicating a solid understanding of the use of controls and other data to explain experimental failures. Our third pedagogical goal is that students master the calculations and techniques involved in the making of complex solutions. Students are required to make their own solutions at multiple points in the laboratory course, including a sevencomponent solution for the final kinetic analysis of their MTHFS mutants. Student mastery of these skills has been assessed through questions about the making of complex solutions on their final exams, in three items across laboratory reports 1 and 4, and on a standardized ACS exam for biochemistry. The instructor-written final exam question (shown in the Supporting Information, p S59) resembles the solutions students make in their final 3 weeks of this course. Some students encounter great difficulty in making these solutions and must repeat their kinetics experiments multiple times. However, by the end of the course, students appear to have mastered this technique, as 88% of them answered this question correctly on the final exam, and those who had errors still achieved 80% or better (n = 24). One question on the ACS exam is related to this skill. Students performed more poorly on this question than on the instructor-written question, with 54% answering correctly, but substantially better than the 43% difficulty index in the national-norm data set (Table 3, question 1). Knowledge of the theory and skill in the practice of common laboratory techniques is the fourth pedagogical goal for this course. Practical skill is assessed by looking for successful completion of each experiment, as described in the laboratory

Roanoke College does not have an Institutional Biosafety Committee, but according to NIH guidelines, none of the work described here exceeds BSL-1.

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RESULTS AND DISCUSSION This laboratory course has been in place for 9 years. Roughly half of the students complete the course without major errors that would prevent moving on to a subsequent step. Others need to be “rescued” with donor material. The most common errors of this type are failure of the site-directed mutagenesis reactions to produce products in the second laboratory period (discovered in the third) and failure to change the codon of the wild-type MTHFS in the second laboratory period (discovered in the fifth). Students encountering these or other catastrophic results are provided with an archived sample from a previous class, a sample from a fellow student, or an archived research22,23 sample. Positive and negative controls are run throughout the course, and these are used to inform technique penalties in grading when failures occur. This course has four main pedagogical goals (Table 1). These goals are assessed through laboratory reports, worksheets, and questions on exams. For laboratory-report items, worksheets, and midterm-exam questions, students are provided with rubric scoring and written feedback on their answers. Written comments and rubric scoring are not provided for final exams. Although some instruments, such as a standardized ACS biochemistry exam, have been administered to all students for many years, other questions have been administered in only some years. Thus, the numbers of students for whom results are reported differs among the items discussed below. Data analysis for this report was approved by the Roanoke College Institutional Research Board, study #18149. Our first pedagogical goal is that students understand how a series of biochemical techniques can be used to test a hypothesis. Most students do successfully collect data that will allow them to test their hypotheses. An example of student results for the K120A mutant is shown in Figure 2. Although this student’s results do not perfectly reproduce the published Km value for ATP for this mutant, they show the expected large increase in the Km compared with that of the wild type, allowing the student to support the hypothesis that the wildtype lysine side chain is critical for ATP binding. When necessary, we try to rescue students with donor material of the same mutant so that they can still test their original hypotheses. (Sometimes a student must “adopt” another student’s hypothesis along with the donor material, depending on what materials are available for rescue.) Students who revert to testing the wild-type enzyme due to having insufficient measurable activity from their mutants can assert that their residue is critical to enzymatic activity in some way, before going on to collect and analyze a full data set on the wild-type enzyme. Thus, even students who have several experimental failures over the course of the semester still come to the end of the semester having conducted a researchlike series of experiments, resulting in the testing of a hypothesis. Student mastery of pedagogical goal 1 is monitored throughout the course. On laboratory reports, students put their laboratory work in context (for example, see report 1, questions 2 and 4, p S46). In some years, students also answer a final-exam question asking them to explain the purpose of each technique used in the laboratory (p S60). For 2017 (n = 14), students averaged 90% of possible points on this exam E



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While MTHFS was chosen as the enzyme to study for this laboratory course, this protocol could be adapted for use with a variety of enzymes for which a straightforward UV−vis assay is available. Of key importance in project selection is that the enzyme can be readily overexpressed and purified, that the enzyme is sufficiently stable to survive student handing, and that the enzyme preparation can be used with minimal loss of activity after several weeks of storage in a laboratory refrigerator or freezer. High-throughput crystallography laboratories can be a good source of plasmids encoding proteins that express well and have known crystal structures. A cocrystal structure with a substrate, substrate analogue, or competitive inhibitor makes it easier for students to formulate hypotheses than a crystal structure without a clearly identified active site.

Table 3. Comparison of Student Success Rates on Selected ACS Exam Questions to Difficulty-Index Values Question

Goals Assesseda

Correct Responses, % (n = 101)

Difficulty Index,b % (n = 465)

1 2 3 4 5 6

3, 4 4 4 4 4 4

54 66 91 91 29 78

43 47 77 80 26 80

a

As in Table 1. bThe difficulty index is the percentage of students answering correctly in the data set used to produce the ACS national norms for this exam.

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reports. Knowledge and practical skill are assessed across 17 laboratory report items, on six ACS-exam questions, and on the midterm and final exams (Table 1). The results from the ACS-exam questions related to laboratory techniques are shown in Table 3. Students performed substantially better on the first four questions than the difficulty index but similarly to the difficulty index for the final two questions. In contrast to questions 1−4, question 5 combines two concepts, one that is not covered in the laboratory and is only sporadically addressed in lecture. Question 6 involves one concept, but it is not consistently taught in the laboratory. These results indicate that pedagogical goal 4 is being met where coverage is complete on a given topic. At multiple points in our biochemistry curriculum, we have found that weaker students have difficulty determining if they are discussing and working with nucleic acids or with proteins, which indicates a deficiency in goal 4. Substantial emphasis is placed on addressing this deficiency in this laboratory course by expecting the correct use of the appropriate term. For example, students are expected to describe changes made early in the laboratory course as changes to the DNA coding for an amino acid as opposed to changes of an amino acid. This expectation is reinforced in the grading of quizzes, exams, and laboratory reports. Students are required to work outside of the traditional laboratory period for several experiments in this semester-long laboratory course. At the outset, there was some concern that there would be substantial student dissatisfaction with this component of the course. In 9 years of student evaluations, there have been very few negative comments about working outside standard laboratory hours. When these comments have occurred, they have been primarily focused on a student’s perception that they were working more than the required 3 h per week, not on when those hours occurred. One motivation for deploying this series of experiments was the possibility of increased student engagement through the results and products impacting future experiments and through the focus being on a medically relevant enzyme. Given the context, with only one small cohort of students enrolled in this laboratory each year, it is not practicable to compare the level of student engagement in the reported semester-long course with a more traditional laboratory schedule that offers a different experiment each week. In general, student comments do indicate enthusiasm for the both the extended project and its medical relevance. In addition to anecdotal benefits to student engagement, the laboratory format also offers benefits for weaker students in its focus on independent solutionpreparation skills.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.8b00028. Laboratory manual and laboratory-report directions, pp S1−S50 (PDF, DOCX) Instructor notes and example assessment questions, pp S51−S63 (PDF, DOCX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Catherine A. Sarisky: 0000-0002-7692-2517 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank our students for helpful feedback during the development of these experiments. We thank the Kim research group for the gift of the pSKB3-MTHFS plasmid used as the wild-type MTHFS template in these experiments and BL21(DE3) cells containing the pSJS1244 helper plasmid used for expression during some offerings of this course.

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REFERENCES

(1) Lewis, J. The Use of Mini-Projects in Preparing Students for Independent Open-Ended Investigative Labwork. Biochem. Educ. 1999, 27 (3), 137−144. (2) Jewett, K.; Sandwick, R. K. Ribose 5-Phosphate Isomerase Investigations for the Undergraduate Biochemistry Laboratory. J. Chem. Educ. 2011, 88 (8), 1170−1174. (3) Witherow, D. S. A Ten-Week Biochemistry Lab Project Studying Wild-Type and Mutant Bacterial Alkaline Phosphatase. Biochem. Mol. Biol. Educ. 2016, 44 (6), 555−564. (4) Craig, P. A. A Project-Oriented Biochemistry Laboratory Course. J. Chem. Educ. 1999, 76 (8), 1130−1135. (5) Farnham, K. R.; Dube, D. H. A Semester-Long Project-Oriented Biochemistry Laboratory Based on Helicobacter pylori Urease. Biochem. Mol. Biol. Educ. 2015, 43 (5), 333−340. (6) Taylor, E. V.; Fortune, J. A.; Drennan, C. L. A Research-Inspired Laboratory Sequence Investigating Acquired Drug Resistance. Biochem. Mol. Biol. Educ. 2010, 38 (4), 247−252. (7) MacDonald, G. Teaching Protein Purification and Characterization Techniques: A Student-Initiated, Project-Oriented Biochemistry Laboratory Course. J. Chem. Educ. 2008, 85 (9), 1250−1252.

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