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Page 1 of 54 Chemistry Education Research and Practice
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3 1 Title: Guided Inquiry in a Biochemistry Laboratory Course Improves Experimental Design Ability
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2 Type: Article
Chemistry Education Research and Practice Accepted Manuscript

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8 3 Running title: Guided Inquiry in Biochemistry Laboratory
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10 4 Authors: Nina M. Goodey*& and Cigdem P. Talgar#&
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12 5 *Department of Chemistry and Biochemistry, Montclair State University, Montclair, New Jersey, 07043
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14 #
6 Center for Advancing Teaching and Learning through Research, Northeastern University, Boston, M.A.
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16 7 02115
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Published on 23 August 2016. Downloaded on 27/08/2016 22:45:05.
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8 Co-corresponding authors
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21 9 Nina M. Goodey
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23 10 Department of Chemistry and Biochemistry
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25 11 Montclair State University
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27 12 1 Normal Avenue
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29 13 Montclair, New Jersey 07043
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32 14 Email: goodeyn@mail.montclair.edu
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34 15 Tel: 973.666.1368 and 973.655.3410; Fax: 973.655.7772
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36 16 Cigdem P. Talgar
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38 17 Center for Advancing Teaching and Learning through Research
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40 18 Northeastern University, 02115
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42 19 360 Huntington Ave.
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45 20 Boston, Massachusetts 02115
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47 21 Email: c.talgar@neu.edu
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49 22 Tel: 617.373.2000; Fax: 617.373.7779
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51 23
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53 24 Keywords: Inquiry laboratory, guided-inquiry, biochemistry education, biochemistry laboratory,
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55 25 undergraduate education, experimental design ability
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26 ABSTRACT
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6 27 Many biochemistry laboratory courses expose students to laboratory techniques through pre-

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8 28 determined experiments in which students follow stepwise protocols provided by the instructor.
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29 This approach fails to provide students sufficient opportunities to practice experimental design
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13 30 and critical thinking. Ten inquiry modules were created for a one-semester undergraduate
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15 31 biochemistry laboratory course; these are freely available on the project website. A slightly
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18 32 modified version of the Experimental Design Ability Test (EDAT) was used to assess the impact
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20 33 of inquiry-based learning on student experimental design ability in four experimental (inquiry)
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22 34 and four control (cookbook) sections. EDAT is a published tool that has been validated for use in
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25 35 undergraduate populations (Sirum, 2011). The results, measured by pre- and post-tests, showed a
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27 36 significant positive impact on the experimental design ability of students in sections that
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37 employed the inquiry approach, when compared to those in control sections that employed the
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32 38 cookbook approach. A follow-up conversation with students in a sequel course suggested that the
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34 39 inquiry-based approach also benefited students by promoting self-directed learning.
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40 INTRODUCTION
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6 41 Students entering the workforce are perceived by employers to be insufficiently prepared in

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8 42 skills including problem solving and critical thinking (Casner-Lotto and Barrington, 2006;
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43 Jaschik, 2015). Many of these skills overlap with science process skills, including the ability to
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13 44 design experiments. Indeed, recommendations of faculty committees on core skills and concepts
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15 45 in biochemistry were summarized in multiple valuable articles (Boyer, 2003; Talgar and Goodey,
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18 46 2015; Tansey et al., 2013; Voet et al., 2003; White et al., 2013) and emphasize the need to move
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20 47 away from content-driven curricula and increase focus on process skills (Coil et al., 2010).
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22 48 Furthermore, a recent survey of research-active biochemistry professionals in both industry and
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25 49 academia indicated that employers feel students in biochemistry laboratory courses must master
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27 50 process skills related to designing experiments in order to be successful in research positions
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51 after graduation (Table 1) (Talgar and Goodey, 2015).
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32 Rank Skill
33 1 A mastery of a limited number of basic math manipulations (solution calculations, dilutions,
34 and serial dilutions).
35 2 Understanding the concepts behind the methods, procedures, and assays one works with.
36 3 The ability to look up information.
37 4 Possessing problem solving skills.
38 5 The ability to learn new methods.
39 Table 1. Survey findings (Goodey and Talgar) indicated that biochemistry experts as a group consider
40 the five skills listed in the table, out of 52 skills in the survey, to be the most important for preparing
41 students for the modern research environment.
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54 In the authors’ opinion, biochemistry laboratory courses are, in many instances, taught in a
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6 55 way that does not provide opportunities for students to practice the above skills. Many research

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8 56 method courses in undergraduate curricula are designed around understanding concepts and thus
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57 address skill #2, but may not focus equally on skills #1, 3, 4, and 5 (Handelsman et al., 2004).
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13 58 Biochemistry laboratory courses often employ a cookbook style approach where students
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15 59 perform pre-defined laboratory exercises and are given very detailed instructions on how to
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18 60 accomplish a particular task or experiment. The step-by-step protocols provided in cookbook

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20 61 style courses can deprive students of the opportunities to develop the key skills that these
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22 62 experimental environments have the potential to foster and which employers actively seek. Yet
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25 63 cookbook-style laboratory modules are ubiquitous in biochemistry laboratory courses, perhaps
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27 64 because they are easily assigned to adjunct or graduate student instructors with less experience to
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65 guide a student-directed experimental design and because faculty may lack the knowhow to
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32 66 prepare the necessary materials for inquiry style labs (Cracolice and Monteyne, 2004).
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34 67 Several studies have indicated that inquiry-based learning in biochemistry increases
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37 68 conceptual understanding and improved student attitudes toward their learning (Bailey, 2009;
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39 69 Basaga et al., 1994; Beck et al., 2014; Goldey et al., 2012; Jensen and Lawson, 2011; Knutson et
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41 70 al., 2011). In such learning environments, research skills are taught through inquiry style and/or
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44 71 project-focused labs, where the process is more generative and students are challenged to
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46 72 integrate and apply their declarative knowledge to develop experimental protocols. By designing
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48 73 experiments, in addition to gaining the conceptual knowledge, students better understand the
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51 74 scientific concepts and the math necessary to set up the design. Furthermore, this approach
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53 75 provides critically important opportunities for students to develop flawed experiments, and then
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76 redesign their protocols after receiving feedback from the instructor.
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77 While the number of studies of well-developed inquiry approaches in biochemistry
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6 78 laboratory courses continues to grow (Kirk et al., 2008; Murthy et al., 2014), the assessment of

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8 79 their efficacy must be improved (Ruiz-Primo et al., 2011). In a literature review of metadata on
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80 inquiry learning, Beck and coworkers emphasize the importance of using published assessment
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13 81 tools and employing control groups when assessing the impacts of inquiry based learning (Beck
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15 82 et al., 2014).
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18 83 One goal of the present article is to contribute to the debate concerning the relative merits
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20 84 of inquiry and cookbook learning in laboratory courses (Ault, 2002, 2004; Cracolice and
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22 85 Monteyne, 2004) by providing meaningful data. Such data may encourage departments to
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25 86 consider constructively aligning their biochemistry laboratory courses with the learning goals
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27 87 discussed above by incorporating opportunities for students to gain research skills in addition to
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88 conceptual knowledge. To further understand the value of inquiry-based learning environments
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32 89 in teaching experimental design, we fundamentally transformed half of the sections of an
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34 90 introductory Biochemistry laboratory course “Experimental Biochemistry I” to more closely
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37 91 simulate the modern research environment. Specifically, the inquiry sections were designed to
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39 92 emphasize research skills, such as critical and creative thinking, and experimental design. We
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41 93 compared changes in students’ experimental design ability in these sections to those of students
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44 94 in sections utilizing more traditional instruction that consisted of pre-designed cookbook
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46 95 experiments in which the experimental protocol was provided by the instructor rather than the
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48 96 students. Three months after the course, we evaluated student responses to questions about their
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51 97 impressions of the class. The evaluations indicated a lasting perceived value of this inquiry-based
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53 98 intervention for students. This work addresses the following questions:
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99 1. Does replacing cookbook type laboratory experiments by inquiry-based modules in a
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6 100 biochemistry laboratory course improve students’ ability to design experiements?

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8 101 2. Do students in inquiry-based sections of Experimental Biochemistry I feel more or less
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102 prepared for a research/project oriented second semester course, Experimental Biochemistry
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13 103 II?
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15 104
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18 105 Based on prior studies, we hypothesized that there would be a larger improvement in
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20 106 students’ experimental design ability in the inquiry sections relative to the cookbook sections,
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22 107 and that students in the inquiry sections of Experimental Biochemistry I would be better prepared
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25 108 for a research/project oriented second semester course.
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27 109
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29 110 METHODS
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32 111 Participants
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112 Participants for the study were recruited from the first semester offering of the Experimental
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37 113 Biochemistry I course offered at Montclair State University (MSU). Students in Experimental
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39 114 Biochemistry I were juniors and seniors majoring in Biochemistry, Chemistry, Molecular
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42 115 Biology, or Biology. All students in the courses were invited to participate in the study with an
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44 116 in-person-plea. Students (n=146) enrolled in the first semester offering of the course in two
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46 117 separate first semesters voluntarily participated in the study in return for extra credit points; all
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49 118 students elected to take the assessment, rather than the provided alternative activity, and received
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51 119 extra credit. Students randomly enrolled into the four different sections (2 inquiry and 2
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120 cookbook). Students were not aware of the different teaching approaches in these sections and
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56 121 the inquiry and cookbook sections were randomly assigned only at the beginning of the semester.
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122 Therefore, students were not pre-screened based on interest. During the study, we did not know
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6 123 whether the cookbook or inquiry approach would prove to be more effective and did not

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8 124 intentionally disadvantage any group. Approval was obtained from the MSU Internal Review
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125 Board for this study (Protocol # 001272). Only the scores from students that had longitudinal
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13 126 data (both pre- and post EDAT test scores) were included in the analysis; the analyzed sample
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15 127 size was 103 students. Some of the students (39) in the study continued into Experimental
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18 128 Biochemistry II during the second semester.

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23 130 Experimental Design
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26 131 In this 2 (pedagogical intervention) X 2 (pre-post test) mixed methods quasi-experimental study,
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29 132 participants were derived from two different iterations of the Experimental Biochemistry I course
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31 133 (Inquiry vs. Cookbook). A total of 39 participants, for whom we had longitudinal data, were in
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33 134 the inquiry-based courses while 64 participants were in the cookbook version. All participants
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36 135 were administered the EDAT within the first two weeks of the course. Both groups were
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38 136 exposed to the same experiments and biochemistry techniques, just in a different educational
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137 format. Two sections each semester used the new, inquiry-based modules and two sections used
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43 138 the cookbook modules. This allowed us to compare results from the control group (cookbook
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45 139 labs) to the group that used the new inquiry modules (Figure 1) and also compare results between
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48 140 the inquiry modules being implemented by different faculty members. Each semester, there were
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50 141 a total of 3 instructors. One instructor (co-author NG) instructed two sections (one inquiry and
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52 142 one cookbook) which the remaining two sections were instructed by two different instructors.
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55 143 One of these sections was inquiry and one was cookbook.
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Fall 1 Fall 2
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5 Experimental Design
6 Ability Test (EDAT)

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Inquiry n = 39
8 Inquiry Instructor A Inquiry Instructor A
9 Cookbook n = 64
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12 Inquiry Instructor B Inquiry Instructor D Pre- and post test
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16 Cookbook Instructor A Cookbook Instructor A
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19 Cookbook Instructor C Cookbook Instructor C
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23 145 Figure 1. A schematic overview of the experimental design. During Fall 1 and Fall 2 there were
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146 two inquiry and two cookbook sections; the instructors are indicated by A, B, C and D. A total
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28 147 of 103 students took both the EDAT pre- and post-tests and their data was included in the
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30 148 analysis.
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149 Design of Inquiry Modules
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35 150 Prior to the first implementation of the Inquiry modules (Fall 1), experiments used at MSU in
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37 151 Experimental Biochemistry I were a set of predefined exercises where students followed step-by-
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40 152 step instructions on how to conduct an experiment. We created a revised set of laboratory
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42 153 modules that were hypothesis driven and focused on fundamental concepts in biochemistry while
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44 154 maintaining the topic matter of the cookbook experiments (Table 2). The modules follow a
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47 155 logical sequence and address different aspects of the same enzyme, dihydrofolate reductase; a
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49 156 sample module is provided in the appendix (Appendix D) and the remaining modules can be
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157 freely downloaded from the project website (http://www.montclair.edu/csam/nsf-tues-grant/).
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54 158 Some of the modules use materials created in previous modules. For example, students purify the
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56 159 protein dihydrofolate reductase and then do kinetic and inhibition studies using their purified
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160 sample in later modules. A laboratory sheet was prepared for each experiment and contained
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6 161 sections shown in Table 3.

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8 162
9 Inquiry Module Title Module Purpose/Goal
10 Module 1: Checking the calibration Design and conduct an experiment to determine the precision and
11 of a micropipette accuracy of a micropipette using the gravimetric method.
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13 Module 2. Determination of Design and conduct an experiment to determine the concentration
14 unknown NADPH concentration (in mM) in the provided unknown NADPH sample.
15 using absorbance
16 spectrophotometry
17 Module 3. Establishing Standard Design and implement a protocol to establish a standard curve for
18 Curve (Bradford Assay) BSA that shows the relationship between absorbance and protein
19 concentration.
20 Module 4. Protein Purification by Separate the protein Dihydrofolate Reductase (DHFR) from a
21 Chromatography mixture of many proteins.
22 Module 5. Determination of Protein Design and conduct an experiment to look at the purity of your
23 Purity by SDS-PAGE DHFR sample and the relationship between amount of protein
24 loaded in a well and the appearance of the resulting band in an
25 SDS-PAGE gel.
26 Module 6. Protein Concentration Design and implement a protocol to establish the standard curve
27 Determination for BSA and determine the concentration of protein in the samples
28 from Module 4.
29 Module 7. Measuring DHFR Design and implement a protocol to examine the effect of DHFR
30 Catalytic Activity – Effect of (enzyme) concentration on the time dependence of the DHFR
31 enzyme concentration of reaction catalyzed reaction. You will also use the data to determine the
32 rate. catalytic activity of DHFR.
33 Module 8. Effect of the DHFR Your assignment is to plan and conduct an experiment to examine
34 inhibitor Trimethoprim on DHFR the effect of TMP (inhibitor) concentration on the time dependence
35 catalytic activity of the DHFR catalyzed reaction. You will determine an IC50 value
36 for the inhibitor.
37 Module 9: Extraction of Plasmid You will receive a frozen pellet of E. coli cells containing a
38 DNA, Restriction Digest, and DNA plasmid. Design and implement a protocol to determine the size
39 Gel Electrophoresis of the plasmid and the insert within the plasmid.
40 Module 10: Bioinformatics Design an in silico protocol to 1.) Determine number of BamHI and
41 Presentations NdeI restriction sites in B. stearothermophilus DHFR DNA plasmid
42 and the size of the insert in base pairs; 2.)Determine the protein
43 sequence of the B. stearothermophilus DHFR; 3.) Predict the
44 molecular weight, number of amino acids, the pI, and molar
45 extinction coefficient of B. stearothermophilus DHFR; 4.)Determine
46 the % identity of B. stearothermophilus DHFR with DHFRs human,
47 B. amyloliquefaciens and G. thermodenitrificans.
48 Groups give presentations on their favorite module
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49 Table 2. 1 Semester Experimental Biochemistry I Inquiry Modules. The goal/purpose of each inquiry-
50 based module is provided on the right. All modules address different aspects of the enzyme
51 Dihydrofolate Reductase.
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3 Order Section in Laboratory Sheet
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1 Introduction to module (in easy to understand terms)
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2 Purpose/goal of the module (See Table 2)
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3 Background on the methods/instruments students might use and references for associated

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readings
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4 A list of the available tools, chemicals, instrumentation and other supplies
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5 Math moment: a set of related lab math exercises that lead students to the right path with the
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6 Vocabulary
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13 7 Advice for experimental design
14 8 Common mistakes (list of common pitfalls while designing/doing the experiment)
15 9 Safety information
16 Table 3. Sections and their order in the laboratory sheets are shown. The modules can be freely
17 downloaded from the project website (http://www.montclair.edu/csam/nsf-tues-grant/).
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22 167 The modules were formatively revised after each semester they were implemented.
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24 168 Before implementation an undergraduate student worker was employed during the summer
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26 169 before the first and second implementation to test the modules and the modules were revised
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29 170 based on student feedback.
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31 171
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33 172 Corresponding Cookbook Modules
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36 173 The cookbook modules followed the same sequence of related experiments as the modules in the
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38 174 inquiry set but, unlike the inquiry modules, the cookbook module documents provided a step-by-
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175 step protocol instead of the “advice on experimental design” section. Students in the cookbook
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43 176 sections did not have to turn in experimental designs as assignments each week. In place of
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45 177 writing an experimental plan, the cookbook cohorts were expected to study the provided
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48 178 experimental protocol before coming to lab. They submitted all other materials including
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50 179 answers to math moment questions and data sheets and reports, just like the inquiry group. All
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52 180 exams used in the different sections were identical.
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183 Implementation of Inquiry Modules
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6 184 Students received the inquiry assignment sheets a week before the laboratory session to allow

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8 185 time for groups (2-3 members per group) to design an experiment (or set of experiments) that
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186 would answer the question posed or accomplish the stated goal (Table 2). Students also
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13 187 completed math moment questions individually before coming to lab. Students then carried out
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15 188 the experiment/s and recorded their process and analyzed data. Time was built into each module
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18 189 for students to fail, receive instructor feedback, understand limitations of experimental methods,
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20 190 and try again without judgment. During the semester, students prepared five data sheets with
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22 191 figures and calculations (one per module for modules 1-3 and 9-10) individually and two
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25 192 comprehensive laboratory reports spanning modules 4-6 and 7-8 in groups. This distribution of
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27 193 individual and group work forced each student to perform calculations, data analysis, and
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194 figure/table preparation individually but also gave students exposure to working on preparing
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32 195 and revising laboratory reports in groups, as they often are likely to do in a work environment.
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34 196 Each group chose one module and presented a 15 minute talk on their approach and findings at
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37 197 the end of the semester. Both experimental and control groups were exposed to the same
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39 198 biochemistry lectures, delivered in traditional style (lecture with occasional group problem
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41 199 solving).
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44 200
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46 201 Experimental Design Ability Test (EDAT)
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48 202 We used the EDAT, which measures students' understanding of the criteria for good
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203 experimental design through open-ended responses to a prompt grounded in an everyday life
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53 204 science problem (Sirum and Humburg, 2001). EDAT was administered for each section of
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55 205 Experimental Biochemistry I at the beginning and end of the course. There were two different
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58 206 scenario versions of the EDAT test. The administration of the test was counterbalanced such that
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207 half of the participants received the “Iron is good for women” version as the pre-test and the
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6 208 “Ginseng” as the post-test and the other half of the participants received the reverse order (See

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8 209 Supplementary Materials for the EDAT tests). The study was originally planned for three years
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210 employing a control group (2 sections) where the cookbook approach was used and an
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13 211 experimental group (2 sections) where the inquiry approach was used. Once we analyzed the
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15 212 data and found that the inquiry approach resulted in improved student learning, we decided to
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18 213 only run inquiry sections during the third year.

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20 214 The EDAT responses were scored based on a rubric provided by the developers of the
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22 215 EDAT test; the prompts provided to students are available for review in Appendix A. The
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25 216 authors needed to make minor revisions to the rubric to clarify grading and better capture the
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27 217 intricacies of experimental design ability in the biochemistry laboratory. These changes are
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218 described in Appendix B and the slightly revised rubric is available for review in Appendix B.
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32 219 Scoring was performed by three independent graders (two faculty members and one graduate
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34 220 assistant). Student responses were provided to the graders as a list of randomized responses,
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37 221 without codes, names, or section information. The graders did not know whether a particular
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39 222 response originated from a student in an inquiry section or a cookbook section and whether the
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41 223 response was written at the beginning or at the end of the semester. All student responses were
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44 224 graded independently by the three different graders and the final scores were an average of these
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46 225 grades. As there is an element of subjectivity in the grading protocol, the graders, to the best of
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48 226 their ability, standardized the grading by discussing the individual grades assigned to a sample
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51 227 set of answers as a group to ensure that they were in agreement on how to apply the rubric.
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53 228 Originally, a grading manual was created, and later on, the original rubric was modified to add
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229 brief instructions for graders on some of the grading items to increase consistency (as described
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6 230 in Appendix B).

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232 Follow-up Conversations
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13 233 Following the completion of the second semester laboratory courses, we had four follow-up
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15 234 conversations with students to better understand students’ perceptions of their preparation for a
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18 235 classroom-based research experience in an advanced Experimental Biochemistry II course. This

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20 236 advanced biochemistry laboratory course met twice a week for three hours and was only required
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22 237 for biochemistry majors. Student groups (2-4 students) were given different mutants of B.
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25 238 stearothermophilus DHFR and a goal for their semester long project. For example, a group was
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27 239 given the goal to determine whether the mutation impacts the catalytic activity of the enzyme.
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240 With instructor support, students designed a sequence of experiments (expression and
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32 241 purification of wildtype and mutant dihydrofolate reductase, protein analysis, and enzyme
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34 242 kinetics) that they conduct in the laboratory. Once they met their first project goal, students came
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37 243 up with a research question and designed more experiments. Students had to do significant
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39 244 problem solving and troubleshooting in the process.
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41 245 During the follow-up conversations, our goal was to gain insight into whether students,
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44 246 who had the inquiry-based experience in Experimental Biochemistry I, felt better prepared for the
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46 247 research experience compared to students who had been in one of the cookbook sections. These
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48 248 groups consisted of 5-8 participants from two sections of the follow-up course Experimental
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51 249 Biochemistry II enrolled during the semester following the Experimental Biochemistry I course.
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53 250 Students were recruited by in-person invitation during class in Experimental Biochemistry II; all
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251 students enrolled in Experimental Biochemistry II elected to participate in the conversations. The
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252 participants were thus selected based on their continuation in Experimental Biochemistry II. As
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6 253 only biochemistry majors are required to take the second course in the sequence, all participants

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8 254 were biochemistry majors at the time of the follow-up conversation. The participants were
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255 grouped as a function of which version of the previous course they took. There were a total of 26
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13 256 students who participated in the sessions: 11 students from previous inquiry and 16 students from
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15 257 previous cookbook sections. Each session (~45 minutes) was facilitated by a skilled moderator
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18 258 who recorded student comments. The facilitator did not know which group (inquiry or
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20 259 cookbook) she was working with at each session. The aims for the follow-up conversations were
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22 260 to obtain feedback on how prepared students felt for the advanced biochemistry laboratory
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25 261 course (Experimental Biochemistry II) and whether this depended on whether a student was
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27 262 enrolled in an inquiry or cookbook style course during the previous semester. We did not
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263 thoroughly quantitate the numbers of times particular issues were mentioned or conduct in depth
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32 264 thematic analysis; consequently, the findings from the follow-up conversations must be
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34 265 interpreted with caution.
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37 266
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39 267 RESULTS AND FINDINGS
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41 268 EDAT Data
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44 269 The mean of the EDAT scores for the inquiry groups increased significantly from 3.8 ± 0.40 to
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46 270 4.9 ± 0.39 while the mean of the EDAT scores for all cookbook students decreased from 5.4 ±
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48 271 0.25 to 5.0 ± 0.25 (Figure 2). Mixed Analysis of Variance revealed an interaction between type
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51 272 of condition (inquiry vs cookbook) and scores pre and post to be significant (F(1,101) = 9.00, p =
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53 273 0.003, d=0.45). These data show that the inquiry approach resulted in higher gains in
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274 experimental design ability than the cookbook approach in our course and in our setting.
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27 276 Figure 2. EDAT pre- and posttest mean scores with standard errors are shown for the inquiry
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29 277 and cookbook sections.
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34 279 The EDAT results were not significantly different between the two Fall semesters when
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36 280 data was obtained. Mixed Analysis of Variance revealed no significant interaction between
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39 281 semester (Fall 1 vs. Fall 2) and pre and post-test EDAT scores. There was also no significant
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41 282 difference in the changes in EDAT scores between the instructors who taught the inquiry
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43 283 courses: Analysis of Variance showed no significant interaction between instructor and pre and
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46 284 post-test EDAT scores. Similarly there was no significant different in the changes (pre- to post-
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48 285 test) in EDAT scores between different instructors who taught the cookbook sections. Using the
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50 286 rubric associated with the EDAT, students’ EDAT responses were independently scored by three
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53 287 raters. The scores were then compared for inter-rater reliability. Each rater was able to score
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55 288 each of the responses that were analyzed and the inter rater reliability was determined to have a
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289 Pearson’s Correlation Coefficient of 0.84 (p < 0.000). This indicates that the three raters’
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6 290 responses were correlated.

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8 291 Figure 3 shows the inquiry- and cookbook section changes in pre- to post-test mean
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292 scores for each individual EDAT rubric item with associated standard errors. The mean changes
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13 293 in pre- to post-test scores for items #1, 2, 3, 5, 7, 8, and 9 were significantly different between
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15 294 the inquiry and cookbook groups; mean values, standard errors, t and p values are listed in Table
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18 295 4. These data suggest that the inquiry approach, compared to the cookbook approach, more
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20 296 effectively prepares students to identify independent and dependent variables (#2 and #5) and
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22 297 understand constants (#7), sample size (#8), and replicates (#9). The absolute inquiry- and
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25 298 cookbook section pre- to post-test mean scores for each individual EDAT rubric item are shown
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27 299 in Appendix C for reference.
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29 Mean Change Pre-to
30 Mean Change Pre-to
Rubric Post Score ± SE
31 Post Score ± SE t-value p-value
Item # (Cookbook sections)
32 (inquiry sections)
33 1 0.13 ± 0.050 -0.03 ± 0.026 2.95 0.004**
34
2 0.14 ± 0.057 -0.06 ± 0.036 3.00 0.003**
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3 0.21 ± 0.060 0.02 ± 0.042 2.53 0.012*
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4 0.21 ± 0.051 0.13 ± 0.040 1.16 0.248
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5 0.07 ± 0.059 -0.11 ± 0.046 2.41 0.017*
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6 0.05 ± 0.046 -0.07 ± 0.043 1.86 0.064
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40 7 0.08 ± 0.049 -0.20 ± 0.045 4.16 0.000**
41 8 0.16 ± 0.055 -0.02 ± 0.041 2.73 0.007**
42 9 0.12 ± 0.035 0.01 ± 0.028 2.36 0.019*
43 10 0.00 ± 0.036 -0.08 ± 0.031 1.64 0.102
44 11 0.07 ± 0.033 -0.02 ± 0.034 1.81 0.071
45 300
46 301 Table 4. The inquiry- and cookbook section changes in pre- to post-test mean scores for each individual
47 302 EDAT rubric item with associated standard errors are shown. The mean changes in pre- to post-test
48 303 scores for items #1, 2, 3, 5, 7, 8, and 9 were significantly different between the inquiry and cookbook
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304 groups (indicated in bold); mean values, standard errors, t – and p values are shown. Items with p ≤ 0.05
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51 305 and p ≤ 0.01 are indicated by * and **, respectively.
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306 To our surprise, for rubric items #5 and #7, there was also a significant pre- to post-test
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6 307 decrease in the cookbook group’s mean scores. For item #5, the mean pre-test score in the

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8 308 cookbook sections (0.73 ± 0.032 (SE)) was significantly higher than the post-test mean score
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309 (0.63 ± 0.035); t = 2.39, p = 0.018. Similarly, within the cookbook sections, for items #7 and
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13 310 10, the mean pre-test scores (0.42 ± 0.036 and 0.16 ± 0.027) were significantly higher compared
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15 311 to the post-test mean scores (0.22 ± 0.030 and 0.083 ± 0.020); t =4.39 and 2.50, p= 0.000 and
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18 312 0.013, respectively, for questions # 7 and #10 (Appendix C).

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4 *
0.25 ** Inquiry
5 Change in Mean Score ** **
6 Cookbook
* ** *

7 0.15
(Pre- to Post-test)
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10 0.05
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12 -0.05
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14 -0.15
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16 Rubric Item #
17 -0.25
18 1 2 3 4 5 6 7 8 9 10 11
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20 1. Recognition that an experiment can be done to test the claim 7. Realization that at least one variable must be held
21 (vs. simply reading the product label) constant (e.g. age)
22 2. Identification of what variable is manipulated (independent 8. Understanding that the larger the sample size or # of
variable is ginseng vs. something else) subjects, the better the data
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24 3. Define how the independent variable will be manipulated
9. Understanding that the experiment needs to be repeated
(describing the dose, amount, etc.)
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26 10. Awareness that one can never prove a hypothesis, that
27 one can never be 100% sure, that there might be another
4. Identifying the negative control group (no iron) or placebo
experiment that could be done that would disprove the
28 (subjects did not know if they were given ginseng or sugar pill)
hypothesis, that there are possible sources of error, that there
29 are limits to generalizing the conclusions
30 5. Identification of the dependent variable (memory, endurance,
31 vs. something else) 11. Mentioning the need for a random sample. That the
32 6. Specific definition of measurement of the dependent variable sample has to be representative of the population through
(using cards/photos/words to test memory or distance a person random sampling consisting of different ages, ethnicities, etc..
33
can run to test endurance)
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35 314
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37 315 Figure 3. Mean EDAT rubric item scores changes (post-test score – pre-test score) are shown
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40 316 for all 11 grading items for students in the inquiry sections (gray bars) and for students in the
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42 317 cookbook sections (white bars). A negative value indicates that the mean score for the group
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44 318 decreased from pre- to post-test. Error bars indicate standard errors. The rubric items are listed
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47 319 in the table below. Items where there is a significant difference (p < 0.05 and p<0.01) between
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49 320 the pre- and post-test mean scores are indicated by stars (*) and double stars (**), respectively.
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54 322
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323 Follow-up Conversations
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6 324 Student retrospective reflections are listed in Table 5 and are divided into two columns,

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8 325 comments from students who had had the cookbook vs. inquiry experience. Anecdotally,
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326 students who benefited from the inquiry sections felt better prepared for the classroom based
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13 327 research course and emphasized that they benefited from learning from their own mistakes.
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15 328 Students who took the cookbook lab felt less well prepared for the research based course but also
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18 329 expressed that it challenged and motivated them more than the previous semester’s course
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20 330 (cookbook experience).
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22 331
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4 Students from Cookbook Sections Said: Students from Inquiry Sections Said:
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6 On How Prepared they Feel: On How Prepared they Feel:

7 Feel that in terms of the present class (Exp. Math modules were very helpful
8 Biochem II), they are left on their own Even general modules were very helpful
9 “It would be nice to have the goal clearer in our Very prepared
10 minds, we did not do the planning enough (in A lot of techniques are repeats we are just now
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Exp. Biochem. II)” getting to the mastery level (in Exp. Biochem. II)
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13 It’s the first time we are doing this, last We liked writing our own procedures
14 semester was more structured
15 Feels like we are “jumping in without our On Learning from Failure in Exp. Biochem I:
16 swimmies” We weren’t sure what we had to do, and
17 Feel insecure if they are doing it right messed up a lot (in Experimental Biochemistry
18 Kept asking “Am I doing this right?” I)

19 Huge learning curve
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On Technique: We learned the hard way
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22 Technique wise they feel they are ready You do it wrong, you learn from your mistakes
23 Now we have a general idea If you get everything up front, you don’t know
24 what you don’t know
25 On Exp. Biochem II Experience: Last semester we had to write our own
26 We are going a little further than last semester protocols
27 Feels like what we are doing is more important
28
More emphasis, more interesting, feels like it
29
30 matters more than going through the
31 procedures like how many drops you can feed
32 on a pen
33 This semester is challenging, you have to
34 decide what procedure to following
35 Before we were told what to do, but now
36 depending on the result you have to decide
37
what to do
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39
40 Table 5. Quotes from students during the follow-up conversations administered during the second
41 semester biochemistry laboratory course in the second semester. The left and right panel show
42 quotes from students who had taken the cookbook version or the inquiry version of the first
43 semester course during the previous fall, respectively. The quotes are divided into topical areas.
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45 332
46 333
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334 DISCUSSION
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6 335 This work is founded on the educational theory, constructive alignment, which is an approach to

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8 336 teaching and learning where a student constructs his or her own knowledge and learning though
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337 a set of appropriately designed learning activities. To accomplish this, the teacher creates a
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13 338 learning environment and learning - and assessment activities that support and align with the
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15 339 intended learning outcomes. Constructive alignment suggests that a course’s outcomes should
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18 340 align with the activities in which students engage and with assessments of student performance
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20 341 (Biggs, 1996; Biggs, 2003). In our courses, we want students to learn research skills that will
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22 342 enable their success in modern research positions, including experimental design, laboratory
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25 343 math skills, data analysis, ability to look up information, understanding of the concepts behind
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27 344 methods, troubleshooting, understanding the way a research project progresses, collaboration,
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345 and oral and written presentation skills. The present study investigated the impact of embedding
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32 346 inquiry-driven activities, in which the students develop their own protocols, on ability to design
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34 347 experiments, as compared to cookbook models.
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37 348 This is, to our knowledge, the first investigation of the impact of inquiry labs in a
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39 349 biochemistry laboratory course on student learning using both a control group and a validated
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41 350 experimental design ability assessment, although a slightly modified version (see Appendix B for
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43
44 351 details). The results of the EDAT assessment demonstrate significant advances in students’
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46 352 experimental design thinking in inquiry sections relative to the cookbook condition. These
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48 353 differences suggest that the inquiry-based educational model better, more tightly, aligns the
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51 354 course activities and some of the desired outcomes, specifically the ability to design experiments,
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53 355 than does the cookbook based model.
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356 As discussed above, the data show that students in the cookbook sections, taught in a
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6 357 traditional way, did not make equally large gains as students in the inquiry sections (Figures 1

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8 358 and 3). Surprisingly, students in the cookbook groups also performed worse in the EDAT after
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359 the course, compared to how they performed before the course. It is possible that weekly
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13 360 exposure to cookbook experiences, where students may not think about experimental design as a
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15 361 part of laboratory experimentation, hinders their ability to perform tasks related to designing
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18 362 experiments such as identifying dependent variables (Revised EDAT Rubric #5), understanding
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20 363 the need for keeping experimental variables (other than the independent variable) constant (#7),
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22 364 and considering experimental limitations (#10). This is perhaps analogous to the finding that
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25 365 student exposure to multiple choice only exams (as compared to a mix of multiple choice with
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27 366 constructed-response questions) presents an obstacle for critical thinking in an introductory
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367 biology course.(Stanger-Hall, 2012) Moreover, the finding that exposure to cookbook-style
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32 368 experiences may decrease students’ ability to design experiments parallels a disturbing findings
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34 369 by Barbera and coworkers where they applied the Colorado CLASS-chem instrument that looks
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37 370 at students’ versus experts’ thinking. The study showed that students, after traditional
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39 371 introductory chemistry courses, tend to think less rather than more like experts compared to
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41 372 before the course (Barbera, 2008); similar observations were made in an introductory physics
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44 373 course (Adams, 2006). These findings, and ours, suggest that aspects of traditional teaching
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46 374 methods can be not only ineffective but also potentially harmful.
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48 375 Retrospective reflections from the subsequent semester provide some hints for why
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51 376 students in the cookbook sections may have become less apt at experimental design during
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53 377 Experimental Biochemistry I. Students reported that the cookbook version of Experimental
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378 Biochemistry I was an unstimulating learning environment compared to the advanced course
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379 Experimental Biochemistry II: “Feels like what we are doing is more important (in the advanced
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6 380 course)”, and “More emphasis, more interesting, feels like it matters more than going through the

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8 381 procedures like how many drops can you feed on a pen” (Table 5). We think it is possible that
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382 this reported lack of intellectual engagement and the rote following of protocols in the cookbook
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13 383 courses caused students to think less like scientists, damaging their ability or willingness to
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15 384 design their own experiments.
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18 385 While students self-selected the section they enrolled in, without knowing that there were
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20 386 different teaching approaches, and while the sections were randomly assigned as inquiry or
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22 387 cookbook in the beginning of the semester, one must be cautious when considering whether
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25 388 students were truly randomly assigned into the sections. The mean pre-course EDAT score of
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27 389 students in the cookbook sections was higher than the mean pre-course EDAT score for students
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390 in the inquiry sections (Figures 1); students in the cookbook sections, on average, thus started at
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32 391 a higher level in experimental design ability compared to students in the inquiry groups. Students
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34 392 in the study were from different majors and it is possible that they chose their section based on
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37 393 their course timetable and that there could be a cohort-bias. To the best of our ability, we
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39 394 addressed this by looking at the change in student performance between pre- and post-
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41 395 assessments to account for the possibility that students in one section were more or less prepared
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44 396 compared to another in the beginning of the course.
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46 397 We sought feedback on the long term benefits of the emphasis on experimental design
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48 398 thinking in the inquiry-based sections three months following the conclusion of the course by
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51 399 way of follow-up conversations conducted with students who had completed those courses and
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53 400 were now enrolled in an advanced experimental biochemistry course. These retrospective
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401 reflections suggested that graduates of the inquiry-based course believed they were “very
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402 prepared (for the advanced course)” (Table 5). These students were able to identify how the
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6 403 advanced course was giving them an opportunity to further master skills they learned in the first

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8 404 course. These students possessed a clear understanding of the role that each course played in
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405 their development “we learned the hard way”, and commented on the “huge learning curve”;
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13 406 they noted that Experimental Biochemistry I provided them a valuable opportunity to become
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15 407 experts in experimental design. On the other hand, those who completed the cookbook version of
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18 408 Experimental Biochemistry I felt under-prepared for Experimental Biochemistry II, students
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20 409 stated that they “felt insecure”, “not sure if they are doing it right”. They discussed how it was
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22 410 the latter course, which instructed them about experimental design for the first time: “It’s the first
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25 411 time we are doing this, last semester was more structured”, “Feels like we are “jumping in
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27 412 without our swimmies”, and “..feel that in terms of the present class (Experimental Biochemistry
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413 II), we are left on our own” (Table 5). These comments underscore how relatively unprepared
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32 414 the cookbook experience left students to tackle their research projects in Experimental
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34 415 Biochemistry II.
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37 416 Lastly, students who had taken the course utilizing inquiry-based instruction, in addition
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39 417 to understanding experimental methodology, reported having acquired skills such as learning
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41 418 from failure (self-directed learning) which was number five on the list of skills that both industry
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44 419 and academic professionals felt were central to students’ preparation (Talgar and Goodey, 2015).
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46 420 Students from inquiry group in the follow-up conversation reflected on Experimental
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48 421 Biochemistry I as follows: “We weren’t sure what we had to do and messed up a lot”, “You do it
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51 422 wrong, you learn from your mistakes”, and “If you get everything up front, you don’t know what
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53 423 you don’t know”. These comments suggest that students from the inquiry sections were able to
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425 design skills and may thus have benefited more from their research project experiences in the
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6 426 advance course. The ability to practice experimental design, over time, in two settings may have

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8 427 consolidated student understanding; it can be useful to look at inquiry experiments in the context
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428 of a curriculum in addition to focusing on individual courses.
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13 429 The finding that the inquiry approach results in students feeling better prepared for a
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15 430 class-based research experience may be particularly significant for those institutions specifically
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18 431 interested in preparing their undergraduates for research. The modules we developed surrounding
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20 432 a common theme, dihydrofolate reductase enzymatic activity and inhibition are available online
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22 433 at http://www.montclair.edu/csam/nsf-tues-grant/. A sample of cookbook and inquiry modules is
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25 434 shown in Appendix D. We expect instructors at other institutions will adopt these modules for
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27 435 their courses. In the modules, we chose to use a single enzyme to (dihydrofolate reductase) to
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436 allow students to see the longitudinal process of a research project. We think that the benefits of
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32 437 using a single enzyme throughout the semester outweighed the disadvantages of being exposed
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34 438 to only one enzyme and one catalytic reaction. As faculty may prefer to use their own favorite
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37 439 enzyme, we are currently preparing a manuscript, which discusses our experience in converting
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39 440 existing cookbook biochemistry labs to inquiry modules. Brickman and coworkers reported
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41 441 higher gains in science literacy skills in inquiry sections compared to traditional sections in a
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44 442 non-majors introductory biology laboratory course but noted that students expressed some
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46 443 resistance to the inquiry approach (Brickman, 2009). We agree that balancing student
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48 444 independent activities with instructor support should be considered when engaging in inquiry
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51 445 teaching (Brown, 2006) and Vygotsky defines the zone of proximal development to be where
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53 446 students are challenged not too much nor too little (Vygotsky, 1978). Accordingly, we have
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447 provided some suggestions to facilitate inquiry-style biochemistry laboratory teaching in Table 6.
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6 Inquiry Lab Possible Challenge Recommendations for Instructor
Feature

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8 Students Due to lack of Explain the value of experimental design ability in
9 design their experience, students may research positions post-graduation.
10 own struggle with Provide support and encouragement; have students do
11 experiments. experimental design, not the first experimental design in class.
12 know where to start, and Offer constructive feedback on students’ experimental
13 feel overwhelmed or plans in office hours or on-line before they are due.
14 discouraged. Direct students to “Advice for experimental design” section
15 in modules for ideas on how to get started.
16 Experiments Students feel worried Explain the professional value of learning troubleshooting.
17 often fail; about “completing the Emphasize the importance of the research process over
18 students must experiment” or running completing a task.

19 redesign and out of time. Students, Have students analyze their data in lab directly after
20 repeat who are used to failure- performing an experiment and provide ample time to
21 experiments. proofed, confirmatory redesign and repeat the experiment during lab; allow
22 experiments, can feel students to reflect before offering feedback.
23 upset when an Encourage students to view instructor as a coach rather
24 experiment fails. than evaluator.
25 Lab focused on Students may question Explain that biochemical research deals with unanswered
26 unanswered what and whether they questions and that it is “normal and exciting” to not know
27 questions are learning because they all the answers.
28 rather than are not used to open- Be open about not knowing an answer but explain how
confirming ended inquiry or the one can proceed to find out answers.
29
known teacher not having all the Listen to student questions with attention.
30
information. answers. It is helpful for instructor to have research experience.
31
Lab reports are Grading can be time For some modules, have students prepare datasheets
32
based on consuming due to non- instead of full reports; employ peer-review, combine
33
individual standard lab reports with multiple, related modules into one lab report and have
34
experimental different experimental students work on select reports in groups.
35
designs. methods. Utilize rubrics that are general enough for different reports.
36
Requires Students do not Emphasize the importance of studying the concepts
37
understanding understand the concept before experimental design.
38
of concept the question is based on Use on-line videos as pre-lab lectures.
39
behind the lab. and struggle with Use a book that provides the theory behind different
40
experimental design. experimental methods (for example, Modern Experimental
41
Biochemistry by Rodney Boyer).
42
Students Different students/groups Provide background information on instruments on the
43
decide what need training on different experimental modules.
44
instrumentation instruments. Prepare videos on different instruments; make these and
45
to use others available on-line; include safety information.
46
Calculations Students do not know Provide sample math problems in module that relate to the
47
are needed for how to perform particular experiment to “prime” students for experimental design.
48
experimental calculations and struggle Consider grading select math problems to ensure students
49
design. with the design. practice.
50
Students’ Students may encounter Provide students a list of common mistakes (pitfalls) in the
51
experimental different experimental module.
52
designs vary. “pitfalls”. Discuss possible challenges individually with different
53 groups as they are getting started.
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Table 6. Select unique features of inquiry style labs, resulting challenges, and recommendations for
55
practice to address these are listed to assist teachers in adopting the inquiry approach in biochemistry
56
laboratory teaching. Some of the “recommendations for practice” apply to more than one challenge.
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450 We hope inquiry learning through our modules improves student gains in other
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6 451 competencies that have an overlap with experimental design ability. We have not yet directly

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8 452 assessed the impact of inquiry labs on the ability to look up information, to problem solve, and to
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453 learn new methods. We plan to next examine the effectiveness of the inquiry approach on gains
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13 454 in laboratory math skills, ranked as the most important skill based on a recent survey of
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15 455 biochemistry professionals (Talgar and Goodey, 2015). Research that employs the same inquiry
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18 456 modules at a range of different institutions is still needed to determine whether the findings can
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20 457 be generalized.
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22 458 The results presented here may encourage skeptical members of the biochemistry
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25 459 teaching community to consider adopting an inquiry style approach to teaching biochemistry
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27 460 laboratory courses, facilitate peer acceptance of faculty members who wish to implement inquiry
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461 style learning, or generate discussion on the pros and cons of inquiry based learning in the
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32 462 biochemistry laboratory setting. This study sets a standard for assessments of inquiry approaches
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34 463 in biochemistry laboratory courses and may encourage other researchers to employ appropriate
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37 464 assessments. Our students in the inquiry style laboratory setting are now required to define
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39 465 questions, design experiments, and recognize that there are many paths to the same goal in
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41 466 research and that considerable analysis and decision making must occur in this process.
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44 467
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468 ACKNOWLEDGEMENTS
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6 469 This work is supported by a grant from the National Science Foundation: NSF-TUES grant

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8 470 (DUE-1245630) titled "Incorporation of Research Skills into the Undergraduate Biochemistry
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471 Curriculum to Create Extraordinary Scientists for the Modern Research Environment” to Nina
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13 472 M. Goodey, PI, Cigdem P. Talgar, Co-PI, James Dyer, Co-PI and John Siekierka, Co-PI. We
14
15 473 thank Dr. Jim Dyer, Dr. John Siekierka, and Dr. Ueli Gubler for serving as excellent instructors
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17
18 474 in the Experimental Biochemistry course and their educational insights. We thank students Diane
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20 475 Hagman and Tiina Sivonen for trialing the inquiry modules during summers 2013 and 2014,
21
22 476 respectively, and their helpful feedback. We are grateful to Dr. Lynn Schneemeyer for her
23
24
25 477 insights for making the inquiry modules successful. We thank Livia Lazzaro for her expert
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27 478 assistance with administering and coding the assessments for this project. We are grateful to Dr.
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479 Adrian Goodey for a critical reading of this manuscript.
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480 REFERENCES
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32 536 excitement and motivation of research to students; Using inquiry and research-based learning in
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34 537 a year-long biochemistry laboratory : Part II-research-based laboratory-a semester-long research
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37 538 approach using malate dehydrogenase as a research model. Biochem Mol Biol Educ 38, 324-329.
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39 539 Murthy, P.P.N., Thomspon, M., and Hungwe, K. (2014). Development of a Semester-Long,
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41 540 Inquiry-Based Laboratory Course in Upper-Level Biochemistry and Molecular Biology. J Chem
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46 542 Ruiz-Primo, M.A., Briggs, D., Iverson, H., Talbot, R., and Shepard, L.A. (2011). Impact of
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48 543 undergraduate science course innovations on learning. Science 331, 1269-1270.
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51 544 Sirum, K., and Humburg, J. (2001). The experimental design ability test (EDAT). Bioscene 37,
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53 545 8-16.
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546 Stanger-Hall, Kathrin F. (2012). Multiple-choice exams: an obstacle for higher-level thinking in
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6 547 introductory science classes. CBE-Life Sciences Education 11.3, 294-306.

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8 548 Talgar, C.P., and Goodey, N.M. (2015). Views from academia and industry on skills needed for
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549 the modern research environment. Biochem Mol Biol Educ 43, 324-332.
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13 550 Tansey, J., Teaster, B.J., Cox, M., Fox, K., Knight, J., Sears, D., and Bell, E. (2013).
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15 551 Foundational Concepts and Underlying Theories for Majors in “Biochemistry and Molecular
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18 552 Biology”. Biochem Mol Biol Educ 41, 289-296.

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20 553 Voet, J.G., Bell, E., Boyer, R., Boyle, J., O'Leary, M., and Zimmerman, J.K. (2003).
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22 554 Recommended Curriculum for a Program in Biochemistry and Molecular Biology. Biochem Mol
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25 555 Biol Educ 31, 161-162.
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27 556 Vygotsky, L. S. (1980). Mind in society: The development of higher psychological processes.
28
29
557 Harvard university press.
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32 558 White, H.B., Benore, M.A., Sumter, T.F., Caldwell, B.D., and Bell, E. (2013). What Skills
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34 559 Should Students of Undergraduate Biochemistry and Molecular Biology Programs Have Upon
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37 560 Graduation? Biochem Mol Biol Educ 41, 297–301.
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39 561
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4 562 Appendix A. Prompts and Grading Rubric for EDAT used in this study.
5 563
6 564 EDAT Prompt 1:

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565
8
9 566 Advertisements for magnesium supplements claim that it promotes endurance. To determine if
10 567 the claim is fraudulent and prior to accepting this claim, what type of evidence would you like to
11 568 see? Provide details of an investigative design.
12
569
13
14 570 EDAT Prompt 2:
15 571
16 572 The claim has been made that women may be able to achieve significant improvements in
17
573 memory by taking iron supplements. To determine if the claim is fraudulent and prior to
18
19 574 accepting this claim, what type of evidence would you like to see? Provide details of an
20 575 investigative design.
21 576
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4 577 Appendix B. Grading Rubric for EDAT used in this study.
5 578
6 579 Note: The rubric was slightly modified from what was published previously (Sirum, 2001). We

7
580 added an item (#3: “Define how the independent variable will be manipulated (describing the
8
9 581 dose, amount, etc.”)) because the ability to consider how to manipulate the independent
10 582 variable is an important skill in biochemistry research. We also added another item (#11:
11 583 “Mentioning the need for a random sample. That the sample has to be representative of the
12
584 population through random sampling consisting of different ages, ethnicities, etc….”). We
13
14 585 removed original rubric item #7 (“Realization that there are many variables that must be held
15 586 constant (vs. only one or no mention”) because it was somewhat repetitive with original rubric
16 587 item #5 (“Realization that there is one other variable that must be held constant (vs. no
17
588 mention)”) and caused confusion between graders. To increase the consistency of grading, we
18
19 589 changed the wording of number original rubric item #4, which is now #6, to be “Specific
20 590 definition of measurement of the dependent variable (using cards/photos/words to test memory).
21 591 Not enough to say conduct a memory test. They must describe some approach to measure
22
592 memory/ endurance”.
23
24 593
25 594
26 595 Rubric: Student response can receive either 1 or 0 points for each item.
27 596
28
29 597 ____ 1. Recognition that an experiment can be done to test the claim (vs. simply reading the
30 598 product label)
31 599 ____ 2. Identification of what variable is manipulated (independent variable is ginseng vs.
32 600 something else)
33
34 601 ____ 3. Define how the independent variable will be manipulated (describing the dose, amount,
35 602 etc.)
36 603 ____ 4. Identifying the negative control group (no iron) or placebo (subjects did not know if they
37 604 were given ginseng or sugar pill)
38
39 605 ____ 5. Identification of the dependent variable (memory, endurance, vs. something else)
40 606 ____ 6. Specific definition of measurement of the dependent variable (using cards/photos/words
41 607 to test memory or distance a person can run to test endurance)
42 608 ____ 7. Realization that at least one variable must be held constant (e.g. age)
43
44 609 ____ 8. Understanding that the larger the sample size or # of subjects, the better the data
45 610 ____ 9. Understanding that the experiment needs to be repeated
46 611 ____ 10. Awareness that one can never prove a hypothesis, that one can never be 100% sure,
47 612 that there might be another experiment that could be done that would disprove the hypothesis,
48
49 613 that there are possible sources of error, that there are limits to generalizing the conclusions
50 614 ____ 11. Mentioning the need for a random sample. That the sample has to be representative
51 615 of the population through random sampling consisting of different ages, ethnicities, etc..
52 616
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54 617
55 618
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4 619 Appendix C.
5 620
6

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622 Supplemental Figure C. Average EDAT rubric item scores (pre- (left bar) and post-test (right
37 623 bar) scores) are shown for all 11 grading items for students in the inquiry sections (diagonal
38 624 lines and black bars) and for students in the cookbook sections (white and dotted bars). Error
39 625 bars indicate standard errors. The rubric items are listed in the table below. Items where there is
40
41
626 a significant difference (P < 0.05) between the pre- and post-test mean scores are indicated by
42 627 stars (*). The data shows that students in general had higher marks for rubric items 1-5
43 628 compared to items 6 – 11. The most challenging items for students were #9 (replication), #10
44 629 (understanding experimental limitations), and #11 (need for a random sample). Item #11 may
45
46
630 not as frequently come up in a biochemistry laboratory setting but replication and experimental
47 631 limitations do.
48 632
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4 633
5 634
6
Appendix D. Sample Inquiry and Cookbook Modules.

7 635
8 636
9
10 637 The remaining modules can be found at the following web address:
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638
13 639
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15 640 http://www.montclair.edu/csam/nsf-tues-grant/
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3 642 Module 7. Measuring DHFR Catalytic Activity – Effect of enzyme concentration of reaction rate.
4
5 643 Inquiry Version.
6 644

7
8
9
10 645 1. Introduction
11
12 646 Enzymes are biocatalysts that speed up chemical reactions. The catalytic activity of an enzyme is the
13 647 number of substrate molecules converted to product per second per 1 molecule of enzyme.
14
15 # 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑡𝑡𝑡𝑡 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝
16 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 (𝑘𝑘𝑘𝑘𝑘𝑘𝑘𝑘) =
17 1 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 ∗ 1 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
18
19 648 Note in the above equation that the units for catalytic activity will be 1/s. You could of course use a
20 649 different unit of time, for example minutes. In this case the units of catalytic activity would be 1/min.
21
22 650 In this module you will determine the catalytic activity for the enzyme DHFR.
23
24
25
651 You will also examine how enzyme concentration affects your results. The enzyme is a catalyst that
26 652 speeds up the process of converting substrate to product. The more enzyme molecules in the reaction
27 653 vessel, the faster the reaction proceeds. This is analogous to a factory: the more employees working, the
28 654 more pieces of leather converted to shoes per hour (or per minute, or per second). The more enzymes
29 655 “working” in your tube, the more molecules of substrate are converted to product per unit time.
30
31 656 To determine the catalytic activity, you will need to know the number of substrate molecules converted to
32 657 product molecules per unit time (see the equation above). To do this, you will mix the enzyme with
33 658 substrate and then see how fast the substrate is converted to product.
34
35
36 659 In a typical experiment, you will measure the change (decrease) in substrate concentration or the increase
37 660 in product concentration over time. In this experiment you will measure the disappearance of substrate
38 661 and cofactor by recording the decrease in Absorbance at 340 nm over time. In your data file, the Y-AXIS
39 662 will be Absorbance340 nm and the X-AXIS will be time. Pay attention to the units of the X-axis, be sure to
40 663 write them down. The slope of the initial linear decrease will represent the change in absorbance over
41 664 time (ΔAbs/min) and can be used to calculate initial velocity (v0). To convert the slope to
42 665 Δ[substrate]/time, you will use a special extinction coefficient (see below).
43
44 666 To obtain the catalytic activity values, you can divide Δ[substrate]/time by the concentration of enzyme in
45
667 the experiment. Just be sure to have the units of enzyme concentration be the same as the units of
46
47 668 substrate concentration so that the units cancel out.
48
49 669 Please bring a memory stick where the measured data can be stored! The data can be
50 670 shared later between all lab members via email or everyone can save the data to their
51 671 own memory stick.
52
53 672
54 673 2. Purpose of the lab
55 674
56 675 Your lab assignment is to run the reaction (DHF + DHFR + NADPH → THF + DHFR + NADP+)
57
676 and monitor the disappearance of the substrate (DHF) and cofactor (NADPH). This data will
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3 677 allow you to determine the catalytic activity of the enzyme you purified. You will also experiment
4
5 678 with different enzyme (DHFR) concentrations to optimize the experiment. DHFR concentration
6 679 is the independent variable, all other parameters are kept constant. The dependent variable will

7 680 be initial velocity.
8 681
9
10 682 Your assignment is to design and implement a protocol to examine the effect of DHFR
11 683 (enzyme) concentration on the time dependence of the DHFR catalyzed reaction. You will
12 684 also use the data to determine the catalytic activity of DHFR.
13 685
14
15 686 3. Agenda for the Day
16 687
17 688 • You must have worked through Math Moment problems before coming to class. Upon
18 689 entry, each student shows their work to instructor to receive points.
19 690 • Instructor presentation on Measuring catalytic activity
20 691 • In class, groups review Math Moment Problems to prepare for experimental design.
21
22
692 • Experimental design: Groups discuss and finalize experimental protocol. Each student
23 693 writes down a step-by-step protocol for the experiment. Note: you must have done most
24 694 of the experimental design before coming to lab.
25 695 • Conduct the experiment in groups. Each student individually records data in their
26 696 personal laboratory notebook.
27 697 • Clean up
28 698
29 699 4. Background
30
700
31
32 701 • Useful information can be found in Chapter 8B. Please, read this section.
33 702 • The enzyme that catalyzes the reaction is DHFR (dihydrofolate reductase) (~18,600 Da).
34 703 DHFR catalyzes the conversion of substrate DHF (dihydrofolate) to product THF
35 704 (tetrahydrofolate).
36 705 • NADPH (nicotinamide adenine dinucleotide phosphate) is the cofactor required for the
37 706 reaction. NADPH donates a hydride (H-) to DHF. NADPH is converted to NADP+ in the
38
707 reaction. The reaction thus has two starting materials (DHF and NADPH) and two
39
40
708 products (THF and NADP+).
41 709 • Detecting the reaction: DHF (substrate) and NADPH (cofactor) absorb more light at 340
42 710 nm than THF (product) and NADP+. Today, the plate reader will be set so that it takes
43 711 many absorbance measurements as the reaction proceeds at specific time intervals.
44 712 This is the “kinetic setting”. The plate reader will only give the time values and the
45 713 absorbance values, you will use Excel to graph and analyze your data.
46 714 • The initial velocity for the DHFR reaction will be determined by measuring the rate of
47 715 enzyme-dependent decrease in absorbance at 340 nm using the extinction coefficient of
48 716 13.2 mM-1 cm-1 and Beer’s law. This means that the values you obtain for the ΔAbs/time
49 717 (using a 0.5 cm pathlength in the microtiter plate well) will be divided by this extinction
50
718 coefficient and pathlength (0.5 cm) as stated in Beer’s law. Note what the units are when
51
52 719 you do this division when extinction coefficient is expressed in units of mM-1*cm-1 
1
53 720 𝑚𝑚𝑚𝑚𝑚𝑚
1
54 � �∗0.5 𝑐𝑐𝑐𝑐
𝑚𝑚𝑚𝑚∗ 𝑐𝑐𝑐𝑐
55 721 • The assay will be conducted in a microtiterplate. The pathlength will be 0.5 cm. Different
56 722 wells will be identical experiments except that each one will have a different enzyme
57
723 concentration.
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724 • Provide your kcat value in units of 1/seconds. Note that the instrument gives the data in
4
5
725 minutes and you will need to do a conversion.
6 726 • Concentrations we will use in the assay (final in microtiterplate well) are NADPH (100

7 727 µM) and DHF (100 µM). A Mastermix is provided that contains buffer, NADPH, and DHF
8 728 at appropriate concentrations.
9 729 • DHF is a suspension and you must extensively mix Mastermix before each time you add
10 730 it to another well.
11 731 • DHF is light sensitive and must be protected from light as much as possible during the
12 732 experiment (use aluminum foil for this purpose).
13 733
14
15 734 5. Math Moment
16 735
17
18 736 1. What three molecules will be needed in the microtiterplate well, in addition to the buffer, to
19 737 start the DHFR catalyzed reaction?
20 738
21 739
22
23 740 2. What are the products of the reaction catalyzed by DHFR?
24 741
25 742
26 743
27
28
744 3. What wavelength must the plate reader be set to in order to monitor the reaction? Do the
29 745 starting materials DHF and NADPH or the products THF and NADP+ absorb more light at this
30 746 wavelength?
31 747
32
33
748
34 749 4. What molecules are present in the beginning of the reaction (time = 0 seconds) in the
35 750 microtiterplate well?
36 751
37
38
752
39 753
40 754 5. Which molecules are being consumed during the reaction? Which molecules are being made
41 755 during the reaction?
42
43
756
44 757
45 758
46 759 6. You will be provided a Mastermix solution containing DHF at 118 µM and NAPDH also at
47
760 118 µM in buffer. If you mix 170 µL of mastermix with 30 µL of enzyme solution in a
48
49 761 microtiterplate well, what is the final concentration of DHF in the well? What is the final
50 762 concentration of NADPH in the well?
51 763
52
764
53
54 765
55 766
56 767 7. Do you expect to see increase or decrease in absorbance at 340 nm as the reaction
57
768 proceeds? Why?
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5 770
6 771 8. What is the extinction coefficient you will use to convert the enzyme-dependent decrease in

7 772 absorbance at 340 nm to change in substrate/cofactor concentration? Include units in your
8 773 answer. See background section.
9
10 774
11 775
12 776 9. We want to measure kcat, which is equal to Vmax/[E]. Explain why in our experiment the
13 777 concentration of the substrate and the cofactor must be much greater than then concentration of
14
15 778 the enzyme (saturating concentrations). Think about the shape of the Michaelis Menten curve.
16 779
17 780
18 781 10. What will happen to the absorbance vs. time slope if you double the amount of enzyme in
19
20 782 the assay?
21 783
22 784
23 785
24
25 786 11. If the initial, linear portion of the line has a slope (Δabsorbance/Δtime x cm) of 0.02/second,
26 787 use the extinction coefficient above to convert this to a slope that gives the Δ[DHF]/Δtime.
27 788 Remember to include units in your answer. Note: you can use Beer’s law in the following way:
28 789
29
30 790 Δ(absorbance) = ε * 0.5 cm * Δ[DHF]
31 791
32 792 Note: Solve for Δ[DHF]!
33 793
34
35
794
36 795 12. If the enzyme concentration in the assay in the question above was 30 nM, what is the
37 796 catalytic activity? Note: you will want to divide the Δ[DHF]/Δtime value by the enzyme
38 797 concentration (see the equation in the introduction). The units of the [DHF] and [DHFR] will
39
40
798 cancel out as long as you make sure that they are in the same units. You can do a unit
41 799 conversion.
42 800
43 801
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45
802
46 803
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3 804 13. Let’s say that you are provided a Mastermix of NADPH and DHF at appropriate
4
5 805 concentrations to get the correct final concentrations where enzyme is saturated with NADPH
6 806 and DHF. You will add 30 µM of enzyme solution to the well for a total concentration of 200 µL.

7 807 You want to make 8 solutions with different concentrations of enzyme. You want each one to be
8 808 a 2-fold dilution of the previous one. Please, write into the table how you will make the
9
10 809 solutions.
11 810
12 Name of Volume of buffer Total volume Final Dilution
13 to add (µL) (µL) Factor
14 D1 100 µL of DHFR 0 100 1-fold (no dilution)
15
stock
16
17 D2 50 µL of D1 50 100 2-fold dilution
18 D3 100
19 D4 100
20 D5 100
21 D6 100
22 D7 100
23 D8 100
24
811
25
26 812
27
28
29
30
31
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35
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3 813 14. See the possible data below. On the graph, label the data 1-6 from the experiment with the
4
5 814 HIGHEST enzyme concentration (data 1) to the lowest enzyme concentration (data 6).
6 815 Remember that the slope of the initial decrease is the initial velocity. Think about what happens

7 816 to the initial velocity as you increase enzyme concentration. Also, label the 3 sets of data that
8 817 would be best for determining the kcat. When you analyze your experimental data for this
9
10 818 module, do it for all the data sets that have a good initial slope and then take the average.
11 819 Remember that you will want to measure the initial velocity (the initial decrease). Once the
12 820 reaction runs out of substrate, there is no longer a decrease and you see a horizontal line
13 821 (plateau). Provide your answer in units of 1/second (note that x-axis here is in minutes). You will
14
15 822 need to do the same conversion for your experimental data.
16 823
17
0.6
18
19
20 0.5
21
Absorbance at 340 nm
22
23 0.4
24
25
26 0.3
27
28
29 0.2
30
31
32 0.1
33
34
35 0
36 0:00:00 0:01:26 0:02:53 0:04:19 0:05:46 0:07:12 0:08:38 0:10:05 0:11:31
37
38 Time in minutes
39 824
40 825
41 826
42 827
43 828
44
45 829
46 830 15. Use the data shown in filled circles in the figure in the question above to determine the kcat
47 831 for the experiment in units of 1/second. Use the extinction coefficient provided in the
48 832 Background Section. Assume that the enzyme concentration in this experiment was 2 nM.
49
50 833
51 834
52 835
53 836
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55 837
56 838
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3 839 16. Below in the graph, draw a reaction curve that you would like to see (Absorbance vs. time).
4
5 840 Now add a curve for what the data would look like if you use way too much enzyme (use thicker
6 841 line). Think about how, in practice, it will take you a few moments to mix your solutions in a

7 842 well, then press the button to start measuring, so your time 0 on the graph is actually a little bit
8 843 after the reaction was initiated. Hint: is it possible that the data looks like a straight line. Where
9
10 844 would the line be? Finally, add a line for what the data would look like if you had way too little
11 845 enzyme (dotted line).
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28 846
29 847 Plot Absorbance (concentration of substrate) vs. time. Label your axes.
30 848
31 849
32
33 850 6. Supplies Provided
34 851
35 852 • Ice buckets and ice
36 853 • Micropipetters and tips
37 854 • Multichannel pipettor
38
855 • 96-well microtiter plates
39
40 856 • Eppendorf tubes
41 857 • plate shaker
42 858 • UV-VIS plate reader
43 859 • Buffer (40 mM HEPES at pH 6.8)
44 860 • Mastermix solution containing DHF and NAPDH both at 118 µM in 40 mM Hepes pH 6.8
45 861 buffer.
46 862 • Enzyme (DHFR)R solution (your purified enzyme from Module 6). Note: it will be
47
863 necessary to convert the enzyme concentration into units of µM.
48
49 864 • Aluminum foil
50 865
51 866 7. Experimental Design
52 867
53 868 • There are three molecules that must be mixed together, along with buffer, to start the
54
869 reaction. In practice you will mix 170 µL of Mastermix with 30 µL of enzyme solution in
55
56
870 the wells.
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871 • You will need to make 8 different dilutions (D1 – D8) of the enzyme solution (2x serial
4
5
872 dilutions (also known as 1:2 serial dilutions)). The first solution (D1) will be the undiluted
6 873 enzyme. For this well, you will simply add 30 µL of the enzyme stock into the well.
•

7 874 When making your 2x serial dilutions of enzyme (D1 – D8), mix each dilution completely
8 875 before making the next dilution.
9 876 • You will need a negative control well where there is no enzyme. In this well, add buffer
10 877 in place of enzyme.
11 878 • In your experiment, you should have 9 wells (8 different enzyme concentrations and one
12 879 negative control).
13
880 • You do not want your reaction to “go” before you are ready to measure so that you will
14
15
881 be able to record the time dependent decrease in absorbance before it is “over”. The
16 882 reaction will not happen until the enzyme is added to the well. For this reason, add
17 883 enzyme at the very last possible moment (when everything else is ready to go and the
18 884 plate is in already in the machine, ready to go in).

19 885 • For the reason above, you should think about the order of your additions to the
20 886 microtiterplate. You should have 9 wells (8 different enzyme concentrations and one
21 887 negative control). You should first add mastermix to all 9 wells. You should then add
22 888 buffer to the negative control well (nothing should happen). Only then should you add
23 889 enzyme to the remaining 8 wells. When adding the different enzyme solutions (D1-D8)
24 890 into the different wells, you should start with the lowest concentration D8 because the
25
891 reaction should be slowest and not likely go to completion before you measure. You
26
27
892 need to complete adding the enzyme solutions into the different wells as quickly as
28 893 possible and then read the plate as quickly as possible so that you do not miss the
29 894 reaction for any of the wells. Be completely prepared before doing this part – you will
30 895 need to think through the steps and practice them with your group. This will be a
31 896 challenging part of the experiment and you will succeed only if you are prepared.
32 897 • One great option for adding the enzyme quickly is to place the different enzyme
33 898 solutions D1 – D8 and buffer for the control into the wells just below the experimental
34 899 wells. Then you can use a multichannel pipettor to transfer 30 µL of the appropriate
35 900 solution to the experimental wells, all at once. This way you will minimize the delay
36 901 between the beginning of the reaction and starting the measurement. The plate can be
37
902 already in the plate reader plate holder when you do this. You might want to practice this
38
39
903 step at your bench with water before you actually do it.
40 904 • This is a delicate reaction. Think what is happening at every step and perform your very
41 905 best to be successful with this experiment.
42 906 • Remember to keep all reagents in the ice. Cover the ice bucket with foil because the
43 907 substrate is sensitive to light. Keep the plate covered with a piece of foil as much as
44 908 possible once the mastermix is in the well.
45 909
46 910
47
48 911 8. Common Mistakes and Some Advice
49 912 • Remember good pipetting technique. Use the correct pipettor (P20, P200, or P1000) for
50 913 the volume you measuring. Please, handle pipettors gently.
51 914 • In solution, DHF is a suspension. If the suspension sits still for a while the solid and
52 915 liquid phases separate. Therefore, before you use the mastermix, mix it by pipetting it up
53 916 and down.
54 917 • DHF is sensitive to light, keep mastermix covered with foil when not using it.
55
918 • MAKE SURE TO LABEL TUBES AND RECORD THE WELLS.
56
57 919 • Be careful with pipetting. If too forcefully pipetted, reagents may splash into other wells.
58 920 • Think in which order to fill the wells.
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921 • Be sure to use a clean tip when necessary. Do not contaminate your samples or
4
5
922 solutions.
6 923

7 924
8 925 9. Vocabulary
9 926
10
927 Catalytic activity, substrate, cofactor, reaction rate, kcat
11
12 928 10. Safety
13 929
14 930 You must wear safety glasses when conducting the experiment. You must never eat or drink in
15 931 the laboratory. You will need to wait in line to use the plate reader. Please, be patient when
16
932 waiting for your turn to use the plate shaker and plate reader. Be gentle with the plate reader
17
18 933 and plate shaker, these are delicate instruments. Any observed violations of these rules will
19 934 result in lower final grade and/or removal from the lab. These safety items are solely the
20 935 responsibility of the student.
21
936
22
23 937 11. Clean up
24 938
25 939 For clean-up, return the remaining original solution to the instructor. Discard dilutions in the sink
26 940 and eppendorf tubes in the regular trash. Do not throw anything in the biohazard waster. Wash
27
28 941 96-well plates and leave them at the sink to dry. Mark the well that you used with a marker.
29 942 Return pipettors in the correct boxes, the last person puts the boxes in the cabinet in the back of
30 943 the lab. Dry your ice buckets and place them back in the cabinet. Place all other items where
31 944 you got them from. Make sure they are clean. Leave your bench ready for the next class to
32
33
945 start working.
34 946
35 947
36 948
37
38
949 12. Homework
39 950
40 951 Data from modules 7 and 8 will combined into a formal lab report (one per group). When
41 952 analyzing data, you should be able to use a few of your slopes (a few wells) to calculate kcat.
42 953 The ones that finished too quickly or too slowly will not be useful. Remember to use the initial
43
44 954 slope (initial linear decrease) to obtain your slope (Δabsorbance/Δtime). Then convert it to
45 955 Δ[DHF]/Δtime. Finally, divide by [E] to determine kcat. Make sure to convert your kcat to units of
46 956 1/seconds.
47 957
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49 958
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3 959 Module 7. Measuring DHFR Catalytic Activity – Effect of enzyme concentration of reaction rate.
4
5 960 Cookbook Version.
6 961 13. Introduction

7
8 962 Enzymes are biocatalysts that speed up chemical reactions. The catalytic activity of an enzyme is the
9 963 number of substrate molecules converted to product per second per 1 molecule of enzyme.
10
11
# 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑡𝑡𝑡𝑡 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝
12 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 (𝑘𝑘𝑘𝑘𝑘𝑘𝑘𝑘) =
13 1 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 ∗ 1 𝑚𝑚𝑚𝑚𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙 𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
14
15 964 Note in the above equation that the units for catalytic activity will be 1/s. You could of course use a
16 965 different unit of time, for example minutes. In this case the units of catalytic activity would be 1/min.
17
18
966 In this module you will determine the catalytic activity for the enzyme DHFR.
19
20
21 967 You will also examine how enzyme concentration affects your results. The enzyme is a catalyst that
22 968 speeds up the process of converting substrate to product. The more enzyme molecules in the reaction
23 969 vessel, the faster the reaction proceeds. This is analogous to a factory: the more employees working, the
24 970 more pieces of leather converted to shoes per hour (or per minute, or per second). The more enzymes
25 971 “working” in your tube, the more molecules of substrate are converted to product per unit time.
26
27 972 To determine the catalytic activity, you will need to know the number of substrate molecules converted to
28 973 product molecules per unit time (see the equation above). To do this, you will mix the enzyme with
29
974 substrate and then see how fast the substrate is converted to product.
30
31
32 975 In a typical experiment, you will measure the change (decrease) in substrate concentration or the increase
33 976 in product concentration over time. In this experiment you will measure the disappearance of substrate
34 977 and cofactor by recording the decrease in Absorbance at 340 nm over time. In your data file, the Y-AXIS
35 978 will be Absorbance340 nm and the X-AXIS will be time. Pay attention to the units of the X-axis, be sure to
36 979 write them down. The slope of the initial linear decrease will represent the change in absorbance over
37 980 time (ΔAbs/min) and can be used to calculate initial velocity (v0). To convert the slope to
38 981 Δ[substrate]/time, you will use a special extinction coefficient (see below).
39
40
41 982 To obtain the catalytic activity values, you can divide Δ[substrate]/time by the concentration of enzyme in
42 983 the experiment. Just be sure to have the units of enzyme concentration be the same as the units of
43 984 substrate concentration so that the units cancel out.
44
45 985 Please bring a memory stick where the measured data can be stored! The data can be
46 986 shared later for each lab member via email or everyone can save the data to their own
47
48
987 memory stick.
49 988
50 989
51 990 14. Purpose of the lab
52 991
53
54 992 Your lab assignment is to run the reaction (DHF + DHFR + NADPH → THF + DHFR + NADP+)
55 993 and monitor the disappearance of the substrate (DHF) and cofactor (NADPH). This data will
56 994 allow you to determine the catalytic activity of the enzyme you purified. You will also do the
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3 995 experiment with different enzyme (DHFR) concentrations to optimize the experiment. DHFR
4
5 996 concentration is the variable, all other parameters are kept constant.
6 997

7 998 Your assignment is to examine the effect of DHFR (enzyme) concentration on the time
8 999 dependence of the DHFR catalyzed reaction. You will also use the data to determine the
9
10 1000 catalytic activity of DHFR.
11 1001
12 1002 15. Agenda for the Day
13 1003
14
1004 • You must have worked through Math Moment problems before coming to class. Upon
15
16 1005 entry, each student shows their work to instructor to receive points.
17 1006 • Instructor presentation on Measuring catalytic activity
18 1007 • In class, groups review Math Moment Problems to prepare for experimental design.
19 1008 • Conduct the experiment in groups. Each student individually records data in their
20 1009 personal laboratory notebook.
21 1010 • Clean up
22 1011
23
24 1012 16. Background
25 1013
26 1014 • Useful information can be found in Chapter 8B. Please, read this section.
27 1015 • The enzyme that catalyzes the reaction is DHFR (dihydrofolate reductase) (~18,600 Da).
28 1016 DHFR catalyzes the conversion of substrate DHF (dihydrofolate) to product THF
29 1017 (tetrahydrofolate).
30
1018 • NADPH (nicotinamide adenine dinucleotide phosphate) is the cofactor required for the
31
32
1019 reaction. NADPH donates a hydride (H-) to DHF. NADPH is converted to NADP+ in the
33 1020 reaction. The reaction thus has two starting materials (DHF and NADPH) and two
34 1021 products (THF and NADP+).
35 1022 • Detecting the reaction: DHF (substrate) and NADPH (cofactor) absorb more light at 340
36 1023 nm than THF (product) and NADP+. Today, the plate reader will be set so that it takes
37 1024 many absorbance measurements as the reaction proceeds at specific time intervals.
38 1025 This is the “kinetic setting”. The plate reader will only give the time values and the
39 1026 absorbance values, you will use Excel to graph and analyze your data.
40 1027 • The initial velocity for the DHFR reaction will be determined by measuring the rate of
41
1028 enzyme-dependent decrease in absorbance at 340 nm using the extinction coefficient of
42
1029 13.2 mM-1 cm-1 and Beer’s law. This means that the values you obtain for the ΔAbs/time
43
44 1030 (using a 0.5 cm pathlength in the microtiter plate well) will be divided by this extinction
45 1031 coefficient and pathlength (0.5 cm) as stated in Beer’s law. Note what the units are when
46 1032 you do this division when extinction coefficient is expressed in units of mM-1*cm-1 
1
47 𝑚𝑚𝑚𝑚𝑚𝑚
48
1033 1
� �∗0.5 𝑐𝑐𝑐𝑐
𝑚𝑚𝑚𝑚∗ 𝑐𝑐𝑐𝑐
49
1034 • The assay will be conducted in a microtiterplate. The pathlength will be 0.5 cm. Different
50
51
1035 wells will be identical experiments except that each one will have a different enzyme
52 1036 concentration.
53 1037 • Provide your kcat value in units of 1/seconds. Note that the instrument give the data in
54 1038 minutes and you will need to do a conversion.
55 1039 • Concentrations we will use in the assay (final in microtiterplate well) are NADPH (100
56 1040 µM) and DHF (100 µM). A Mastermix is provided that contains buffer, NADPH, and DHF
57 1041 at appropriate concentrations.
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1042 • DHF is a suspension and you must extensively mix Mastermix before each time you add
4
5
1043 it to another well.
6 1044 • DHF is light sensitive and must be protected from light as much as possible during the

7 1045 experiment (use aluminum foil for this purpose).
8 1046 17. Math Moment
9 1047
10 1048 1. What three molecules will be needed in the microtiterplate well, in addition to the buffer, to
11
1049 start the DHFR catalyzed reaction?
12
13 1050
14 1051
15 1052 2. What are the products of the reaction catalyzed by DHFR?
16
1053
17
18 1054
19 1055
20 1056 3. What wavelength must the plate reader be set to in order to monitor the reaction? Do the
21 1057 starting materials DHF and NADPH or the products THF and NADP+ absorb more light at this
22
23 1058 wavelength?
24 1059
25 1060
26 1061
27
28 1062 4. What molecules are present in the beginning of the reaction (time = 0 seconds) in the
29 1063 microtiterplate well?
30 1064
31 1065
32
33 1066
34 1067 5. Which molecules are being consumed during the reaction? Which molecules are being made
35 1068 during the reaction?
36 1069
37
38 1070
39 1071
40 1072 6. You will be provided a Mastermix solution containing DHF at 118 µM and NAPDH also at
41 1073 118 µM in buffer. If you mix 170 µL of mastermix with 30 µL of enzyme solution in a
42
43 1074 microtiterplate well, what is the final concentration of DHF in the well? What is the final
44 1075 concentration of NADPH in the well?
45 1076
46 1077
47
48 1078
49 1079
50 1080 7. Do you expect to see increase or decrease in absorbance at 340 nm as the reaction
51 1081 proceeds? Why?
52
53 1082
54 1083
55 1084
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3 1085 8. What is the extinction coefficient you will use to convert the enzyme-dependent decrease in
4
5 1086 absorbance at 340 nm to change in substrate/cofactor concentration? Include units in your
6 1087 answer. See background section.

7 1088
8 1089
9
10 1090 9. We want to measure kcat, which is equal to Vmax/[E]. Explain why in our experiment the
11 1091 concentration of the substrate and the cofactor must be much greater than then concentration of
12 1092 the enzyme (saturating concentrations). Think about the shape of the Michaelis Menten curve.
13 1093
14
15 1094
16 1095
17 1096 10. What will happen to the absorbance vs. time slope if you double the amount of enzyme in
18 1097 the assay?

19
20 1098
21 1099
22 1100
23 1101
24
25 1102 11. If the initial, linear portion of the line has a slope (Δabsorbance/Δtime x cm) of 0.02/second,
26 1103 use the extinction coefficient above to convert this to a slope that gives the Δ[DHF]/Δtime.
27 1104 Remember to include units in your answer. Note: you can use Beer’s law in the following way:
28 1105
29
30 1106 Δ(absorbance) = ε * 0.5 cm * Δ[DHF]
31 1107
32 1108 Note: Solve for Δ[DHF]!
33 1109
34
35
1110
36 1111
37 1112
38 1113 12. If the enzyme concentration in the assay in the question above was 30 nM, what is the
39
40
1114 catalytic activity? Note: you will want to divide the Δ[DHF]/Δtime value by the enzyme
41 1115 concentration (see the equation in the introduction). The units of the [DHF] and [DHFR] will
42 1116 cancel out as long as you make sure that they are in the same units. You can do an unit
43 1117 conversion.
44
45
1118
46 1119
47 1120
48 1121
49
50
1122
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3 1123 13. Let’s say that you are provided a Mastermix of NADPH and DHF at appropriate
4
5 1124 concentrations to get the correct final concentrations where enzyme is saturated with NADPH
6 1125 and DHF. You will add 30 µM of enzyme solution to the well for a total concentration of 200 µL.

7 1126 You want to make 8 solutions with different concentrations of enzyme. You want each one to be
8 1127 a 2-fold dilution of the previous one. Please, write into the table how you will make the
9
10 1128 solutions.
11 1129
12 Name of Volume of buffer Total volume Final Dilution
13 to add (µL) (µL) Factor
14 D1 100 µL of DHFR 0 100 1-fold (no dilution)
15
stock
16
17 D2 50 µL of D1 50 100 2-fold dilution
18 D3 100
19 D4 100
20 D5 100
21 D6 100
22 D7 100
23 D8 100
24
1130
25
26 1131
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3 1132 14. See the possible data below. On the graph, label the data 1-6 from the experiment with the
4
5 1133 HIGHEST enzyme concentration (data 1) to the lowest enzyme concentration (data 6).
6 1134 Remember that the slope of the initial decrease is the initial velocity. Think about what happens

7 1135 to the initial velocity as you increase enzyme concentration. Also, label the 3 sets of data that
8 1136 would be best for determining the kcat. When you analyze you data experimental data for this
9
10 1137 module, do it for all the data sets that have a good initial slope and then take the average.
11 1138 Remember that you will want to measure the initial velocity (the initial decrease). Once the
12 1139 reaction runs out of substrate, there is no longer decrease and you see a horizontal line
13 1140 (plateau). Provide your answer in units of 1/second (note that x-axis here is in minutes). You will
14
15 1141 need to do the same conversion for your experimental data.
16 1142
17
0.6
18
19
20
21
0.5
Absorbance at 340 nm
22
23
24 0.4
25
26
27 0.3
28
29
30 0.2
31
32
33 0.1
34
35
36 0
37 0:00:00 0:01:26 0:02:53 0:04:19 0:05:46 0:07:12 0:08:38 0:10:05 0:11:31
38
39
Time in minutes
40 1143
41 1144
42 1145
43 1146
44 1147
45
46 1148
47 1149 15. Use the data shown in filled circles in the figure in the question above to determine the kcat
48 1150 for the experiment in units of 1/second. Use the extinction coefficient provided in the
49 1151 Background Section. Assume that the enzyme concentration in this experiment was 2 nM.
50
51 1152
52 1153
53 1154
54 1155
55
56 1156
57
58
59
60 51
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DOI: 10.1039/C6RP00142D
1
2
3 1157 16. Below in the graph, draw a reaction curve that you would like to see (Absorbance vs. time).
4
5 1158 Now add a curve for what the data would look like if you use way too much enzyme (use thicker
6 1159 line). Think about how, in practice, it will take you a few moments to mix your solutions in a

7 1160 well, then press the button to start measuring, so your time 0 on the graph is actually a little bit
8 1161 after the reaction was initiated. Hint: is it possible that the data looks like a straight line. Where
9
10 1162 would the line be? Finally, add a line for what the data would look like if you had way too little
11 1163 enzyme (dotted line).
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28 1164
29 1165 Plot Absorbance (concentration of substrate) vs. time. Label your axes.
30 1166
31 1167
32
33 1168
34 1169 18. Supplies Provided
35 1170
36 1171 • Ice buckets and ice
37 1172 • Micropipetters and tips
38
39
1173 • Multichannel pipettor
40 1174 • 96-well microtiter plates
41 1175 • Eppendorf tubes
42 1176 • plate shaker
43 1177 • UV-VIS plate reader
44 1178 • Buffer (40 mM HEPES at pH 6.8)
45
1179 • Mastermix solution containing DHF and NAPDH both at 118 µM in 40 mM Hepes pH 6.8
46
47
1180 buffer.
48 1181 • Enzyme (DHFR)R solution (the purified enzyme from Module 6). Note: it will be
49 1182 necessary to convert the enzyme concentration into units of µM.
50 1183 • Aluminum foil
51 1184
52 1185
53
54
55
56
57
58
59
60 52
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DOI: 10.1039/C6RP00142D
1
2
3 1186 19. Experimental Protocol
4
1187
5
6 1188 1. Label 8 tubes D1 – D8.

7 1189
8 1190 2. Make serial dilutions of the DHFR enzyme stock in small Eppendorf tubes as shown in the
9
1191 table below. Mix each dilution vigorously before making the next dilution. For example, mix D2
10
11 1192 vigorously before making D3. Fill in the final concentrations in the table below (they are based
12 1193 on stock concentration of enzyme).
13 Dilution Volume of buffer Total volume Final concentration
14 to add (µL) (µL)
15
D1 100 µL of DHFR 0 100
16
17
stock
18 D2 50 µL of D1 50 100
19 D3 50 µL of D2 50 100
20 D4 50 µL of D3 50 100
21 D5 50 µL of D4 50 100
22 D6 50 µL of D5 50 100
23 D7 50 µL of D6 50 100
24
D8 50 µL of D7 50 100
25
26
1194
27 1195 3. Mix the MASTERMIX (provided) vigorously. You must mix MASTERMIX each time before
28 1196 you pipet from it. Place 170 uL of MASTERMIX in wells A1 – A9 in a microtiter plate. Keep
29 1197 plate covered with foil (DHF is sensitive to light).
30
31
1198
32 1199 4. Add 30 µL of buffer to well A9. This is your “no enzyme” control. Since there is no enzyme in
33 1200 well A9, do you expect to see any change in Absorbance over time?
34 1201
35
36
1202 5. Add 50 µL solution D8 to well B8. (Note, not the experiment well A8 but next to it!)
37 1203 Add 50 µL solution D7 to well B7.
40
41
1206 Add 50 µL solution D4 to well B4.
45
1210
46
47 1211 6. You must do the next steps (up to when you hit read on the plate reader) as quickly as you
48 1212 can because the reaction will start when you add enzyme (DHFR). So go to the instrument,
49 1213 make sure all settings are ready to go. Then place plate on the tray. Only then do the following.
50
1214 Use a multichannel pipettor to transfer 30 µL of solutions from wells B1-B8 to wells A1-A8.
51
52 1215
53 1216 7. Read the plate (10 minutes).
54 1217
55
1218 8. Look at your data. Be prepared to repeat the experiment if necessary.
56
57 1219
58
59
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DOI: 10.1039/C6RP00142D
1
2
3 1220 20. Common Mistakes and Some Advice
4
5
1221 • Remember good pipetting technique. Use the correct pipettor (P20, P200, or P1000) for
6 1222 the volume you measuring. Please, handle pipettors gently.
•

7 1223 In solution, DHF is a suspension. If the suspension is left sitting for a while the solid and
8 1224 liquid phases separate. Therefore, before you use the mastermix, mix it by pipetting it up
9 1225 and down.
10 1226 • DHF is sensitive to light, keep mastermix covered with foil when not using it.
11 1227 • MAKE SURE TO LABEL TUBES AND RECORD THE WELLS.
12 1228 • Be careful with pipetting. If too forcefully pipetted, reagents may splash into other wells.
13
14
1229 • Think in which order to fill the wells.
15 1230
16 1231 21. Vocabulary
17 1232
18 1233 Catalytic activity, substrate, cofactor, reaction rate, Kcat

19
1234
20
21 1235 22. Safety
22 1236
23 1237 You must wear safety glasses when conducting the experiment. You must never eat or drink in
24 1238 the laboratory. You will need to wait in line to use the plate reader. Please, be patient when
25
1239 waiting for your turn to use the plate shaker and plate reader. Be gentle with the plate reader
26
27 1240 and plate shaker, these are delicate instruments. Any observed violations of these rules will
28 1241 result in lower final grade and/or removal from the lab. These safety items are solely the
29 1242 responsibility of the student.
30
1243
31
32 1244 23. Clean up
33 1245
34 1246 For clean-up, return the remaining original solution to the instructor. Discard dilutions in the sink
35 1247 and eppendorf tubes in the regular trash. Do not throw anything in the biohazard waster. Wash
36
37 1248 96-well plates and leave them at the sink to dry. Mark the well that you used with a marker.
38 1249 Return pipettors in the correct boxes, the last person puts the boxes in the cabinet in the back of
39 1250 the lab. Dry your ice buckets and place them back in the cabinet. Place all other items where
40 1251 you got them from. Make sure they are clean. Leave your bench ready for the next class to
41
42
1252 start working.
43 1253
44 1254
45 1255 24. Homework
46 1256
47
48 1257 Data from modules 7 and 8 will combined into a formal lab report (one per group). When
49 1258 analyzing data, you should be able to use a few of your slopes (a few wells) to calculate kcat.
50 1259 The ones that finished too quickly or too slowly will not be useful. Remember to use the initial
51 1260 slope (initial linear decrease) to obtain your slope (Δabsorbance/Δtime). Then convert it to
52
53 1261 Δ[DHF]/Δtime. Finally, divide by [E] to determine kcat.
54 1262
55 1263
56 1264
57
58
59
60 54

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18 60 accomplish a particular task or experiment. The step-by-step protocols provided in cookbook

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18 128 Biochemistry II during the second semester.

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18 213 only run inquiry sections during the third year.

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18 235 classroom-based research experience in an advanced Experimental Biochemistry II course. This

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18 312 0.013, respectively, for questions # 7 and #10 (Appendix C).

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18 Kept asking “Am I doing this right?” I)

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18 students must experiment” or running completing a task.

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18 552 Biology”. Biochem Mol Biol Educ 41, 289-296.

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18 884 plate is in already in the machine, ready to go in).

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