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Using Bioinformatics & Molecular Visualization to Develop Student Hypotheses in a Malate Dehydrogenase Oriented CURE

Tamara Mans1, Kevin Callahan2, Jing Zhang3, Ellis Bell4, Jessica Bell4* Affiliations:

1North Hennepin Community College. 2St John Fisher College 3University of Nebraska, Lincoln 4University of San Diego

* Corresponding Author, Department of Chemistry and Biochemistry, University of San Diego, 5995 Alcala Park, San Diego, 92110. Email: [email protected]

Type of Manuscript: CourseSource Lesson Manuscript

Funding and Conflict of Interest: [This work is supported by a grant from the National Science Foundation. NSF-1726932 EHR-IUSE, Principal Investigator Ellis Bell, Co Investigators Jessica Bell and Joseph Provost None of the authors have a financial, personal, or professional conflict of interest related to this work. Copyright: We affirm that we either own the copyright to or have received written permission to use the text, figures, tables, artwork, abstract, summaries and supplementary materials. Title and Description of Primary Image: Mind Map of the Hypothesis Development and Proposal Module

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Abstract

Developing student creativity and ability to develop a testable hypothesis represents a significant challenge in most laboratory courses. This lesson demonstrates how students use facets of molecular evolution and bioinformatics approaches involving protein sequence alignments (Clustal Omega, Uniprot) and 3D structure visualization (Pymol, JMol, Chimera) and an analysis of pertinent background literature to construct a novel hypothesis and develop a research proposal to explore their hypothesis. We have used this approach in a variety of institutional contexts ( Community College, Research Intensive Universities and PUIs ) as the first component in a protein centric CURE sequence built around the enzyme Malate Dehydrogenase that illustrates a variety of foundational concepts from the learning framework for Biochemistry and Molecular Biology. The lesson has three specific learning goals: i) Find, use and present relevant primary literature, protein sequences, structures, and analyses resulting from the use of bioinformatics tools ii) Understand the various roles that non-covalent interactions may play in the structure and function of an enzyme. iii) Create/develop a testable and falsifiable hypothesis and appropriate experiments to interrogate the hypothesis. For each we have developed specific assessment rubrics. Depending on the needs of the course this approach builds to an in class student presentation and or a written research proposal. The module can be extended over several lecture and lab periods. Furthermore, the module lends itself to additional assessments including oral presentation, research proposal writing and the validated pre-post Experimental Design Ability Test (EDAT). Although presented in the context of course based research on Malate Dehydrogenase, the approach and materials presented are readily adaptable to any protein of interest.

1 SCIENTIFIC TEACHING CONTEXT 2 Learning Goal(s) 3 When students have completed this lesson they should be able to:

4 i) Find, use and present relevant primary literature, protein sequences and structures, and 5 bioinformatics tools.

6 ii) Understand the various roles that non-covalent interactions may play in the structure and 7 function of an enzyme.

8 &

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9 iii) Create/develop a testable and falsifiable hypothesis and propose appropriate experiments to 10 interrogate the hypothesis.

11 **Please also list any society generated learning goals that align with your lesson

12 Our three major learning goals encompass several of the stated learning goals (italicized) of both 13 the Bioinformatics and Biochemistry and Molecular Biology frameworks, and associated 14 learning objectives (indented) as detailed below:

15 Bioinformatics:

16 What is the role of computation in hypothesis driven discovery processes within the life sciences

17 Explain the necessity for computation in life sciences research. Explain the role of wet- 18 bench techniques in verifying computational results in life science research. Compare and 19 contrast computer-based research with wet-lab research. Read a scientific article and 20 evaluate how bioinformatics methods were employed by the authors to explore a 21 particular hypothesis. Given a scientific question, develop a hypothesis and define 22 computational approaches that could be used to explore the hypothesis

23 Where are data about the proteome found (e.g., amino acid sequence and structure) and how are 24 they stored and accessed

25 Describe the types of metadata that accompany sequence, structure, and function data to 26 make for useful biological interpretation (e.g. biological source, accession number, 27 UniProt number, journal articles, etc.)

28 How can bioinformatics tools be employed to examine protein structure and function?

29 Query a dataset with a specific protein sequence to learn about potential functions (e.g. 30 Pfam, CDD, SwissProt, UniProt, etc.). View and interpret the structure output from 31 Protein Data Bank (e.g. Cn3D, Jmol, etc.). Propose potential functions for a given protein 32 structure.

33 Biochemistry and Molecular Biology:

34 How do enzymes catalyze biological reactions?

35 Identify the factors contributing to the activation energy of a reaction, Use kinetic 36 parameters to compare enzymes, Interpret the physical meaning of various kinetic

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37 parameters and describe the underlaying assumptions and conditions on which different 38 parameters depend

39 What factors determine structure?

40 Compare and contrast the primary, secondary, tertiary and quaternary structure of 41 proteins, Use bioinformatics and computational approaches to compare primary 42 sequences and identify the impact of conservation and/or evolutionary change on 43 structure and function.

44 How are structure and function related? What is the role of noncovalent intermolecular 45 interactions?

46 Predict the biological and chemical effects of mutation on the affinity of binding and 47 design appropriate experiments to test the predictions

48 What is the Scientific Process?

49 When presented with an observation develop a testable and falsifiable hypothesis, 50 identify appropriate experimental observations and controllable variables

51 What skills are needed to access, comprehend and communicate science?

52 Identify, locate and use the primary literature, Use data bases and bioinformatics tools, 53 Use verbal and visual tools to explain concepts and data.

54 Learning Objective(s) 55 For each of our learning goals we have specific learning objectives with associated rubrics 56 (supplementary material S1) for assessment.

57 Learning Goal 1: Find, use and present relevant primary literature, protein sequences and 58 structures, and analyses obtained using bioinformatics tools

59 Students will be able to:

60 1. Find and use appropriate literature to illustrate the generalizable (big picture) aspects of 61 the work. 62 2. Use appropriate literature to document specific background to the enzyme 63 3. Use appropriate data bases to obtain sequence information, utilize Clustal Omega, 64 Uniprot etc and interpret the resultant data 65 4. Use the protein data base to obtain 3D coordinates for a protein and Pymol or other 66 visualization tools to illustrate key features of the protein and their hypothesis

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67 5. Use appropriate bioinformatics tools to design primers for mutagenesis 68

69 Learning Goal 2: Understand the various roles that non-covalent interactions may play in the 70 structure and function of an enzyme.

71 Students will be able to: 72 1. Compare and contrast the physical basis for coulombic (ionic) interactions and 73 Hydrophobic interactions. 74 2. Outline the types of non covalent interactions you would expect to stabilize 75 secondary structure in a protein 76 3. Outline the types of non covalent interactions you would expect to be involved in 77 maintaining a functional tertiary structure in a protein 78 4. Describe types of non-covalent interactions that might be found in across a subunit 79 interface and the functions of the interactions 80 5. Predict types of non-covalent interactions that would be involved in substrate- 81 enzyme binding and compare their relative strengths 82 6. Describe how non covalent interactions in a protein-substrate complex might 83 promote catalysis 84 7. Predict what types of mutations at nearby residues might alter the pKa of a 85 protonatable group on a protein 86 87 Learning Goal 3 Create/develop and present a testable and falsifiable hypothesis and 88 appropriate experiments to interrogate the hypothesis.

89 Students will be able to:

90 1. Describe how the work fits into the field/fills a gap in knowledge 91 2. Clearly state their hypothesis and the requisite background information that lead to the 92 hypothesis 93 3. Clearly indicate the testable and falsifiable predictions the hypothesis makes 94 4. Briefly outline the types of experiments that will be used to interrogate the hypothesis 95

96 INTRODUCTION 97 The “Vision & Change in Undergraduate Biology Education: A Call for Action” initiative and its 98 following report (1), building on prior publications over the past 15-20 years (2-5) recommends 99 1) integration of core concepts and skills throughout the curriculum, 2) a focus on student- 100 centered learning environments, and 3) validated high impact practices, such as research 101 experiences, as integral components of biology education for all students. Research experiences 102 have major effects on persistence in science (6-9) and positive outcomes in conceptual

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103 understanding and skills development, essential for effective workforce development (10-16). 104 The Council on Undergraduate Research (CUR) defines undergraduate research as inquiry or 105 investigation conducted by undergraduates that makes original intellectual or creative 106 contributions to the discipline (16). Such work is a high impact practice that provides robust 107 learning for students, increases retention, enhances student learning though mentorship by 108 faculty and develops a deeper critical thinking ability, as well as intellectual independence. 109 Realization that authentic research was important for student development was evident in the 110 Boyer Report [17], where smaller schools that provide research mentoring disproportionately 111 produce more graduate school students than research intensive institutions where resources and 112 time allocated to research training are not as focused on undergraduates.. 113

114 Students involved in course-based research [18-24] show that, independent of the type of 115 institution, authentic research experience enhances knowledge gains. It is also important to 116 recognize that while reform to include CUREs in many laboratory courses has been impressive, 117 there remain several important unanswered questions. Assessment has primarily focused on 118 motivation and retention of students (all very positive) but assessment tools linked to student 119 learning outcomes are limited. Further, Brownell and Kloser warn, "despite published articles on 120 CUREs, the impact of these CUREs are still in question" [25]. There have been a number of 121 publications discussing the key elements of a CURE as developed by Lopatto, Dolan, Kloser and 122 others [26-30]. The NSF-funded CURE network (CUREnet) described the key elements of a 123 CURE [19,31]. These elements include: 1) the use of scientific practices, 2) discovery, 3) 124 broadly relevant or important work, 4) collaboration, and 5) iteration. Lopatto and others 125 describe seven components of authentic research [11,12,32] as: novel questions, student- 126 generated questions, development of a hypothesis, experimental design, data collection, data 127 analysis and presentation or publication of the research. Key parameters for such an experience 128 include: minimized role of instructor, an unknown scientific outcome, a project of student design 129 where students are responsible for the design and do most of the work. 130 131 In the current lesson we have built on previously published work using a CURE based on Malate 132 Dehydrogenase (33), and incorporated a number of foundational concepts and skills (3, 34-36)

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133 that are taken from the ASBMB approved Biochemistry and Molecular Biology Learning 134 Framework (37). 135 In many CURE type activities the emphasis is on data collection and analysis. Here we place the 136 emphasis of Hypothesis development and proposal construction, areas that are frequently 137 difficult to incorporate into a CURE or coursework in a meaningful way (38-44). We have also 138 placed a focus on collaboration and teamwork since both contribute to the high impact nature of 139 the lesson. Many CUREs use molecular biology techniques and answer other types of questions, 140 here we place emphasis on protein structure-function relationships, and in particular the role that 141 non covalent interactions (a gateway concept in biochemistry) (46) play in enzyme function.

142 While, as described here, the focus of the lessons presented is on structure-function relationships 143 in Malate Dehydrogenase, the approach, materials and rubrics we have developed are broadly 144 applicable to almost any protein of interest and can be easily tailored to a faculty person’s given 145 research interests.

146

147 Intended Audience 148 This lesson is intended for a broad audience and has been used with a variety of students ranging 149 from incoming first year students in a PUI, 1st & 2nd year Community College Students, and 150 junior/senior biochemistry majors in both PUI and Research Intensive University settings. It has 151 also been used with 3rd/4th year Non Science Majors.

152 Required Learning Time 153 The lesson has been implemented in a variety of formats using up to a total of 8 teaching 154 sessions (for example 4 lecture periods plus 4 laboratory sessions.) Students have multiple 155 homework assignments associated with the lesson and are expected to spend about 4-5 hours out 156 of class working on the lesson.

157 Pre-requisite Student Knowledge 158 For all, not only upper level majors, the lesson requires a basic knowledge of the central dogma, 159 an introductory knowledge of the levels of protein structure (primary, secondary, tertiary & 160 quaternary) and function (catalytic mechanism and enzyme kinetics) and an understanding of 161 various types of non-covalent interactions.

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162 Pre-requisite Teacher Knowledge 163 A basic understanding of protein structure and function, basic enzymology and bioinformatics as 164 well as fundamentals of evolution and molecular biology, although these topics are covered in 165 the two assigned readings associated with the lesson.

166

167 SCIENTIFIC TEACHING THEMES 168 Active Learning 169 Think-Pair-Share

170 Group Work

171 Team work

172 Oral Presentations

173 Mind Map Creation

174 Journal Club Discussion

175 Assessment 176 Homework

177 In Class Quiz

178 Oral Presentations and Worksheets

179 Written Presentation

180 Inclusive Teaching 181 The various components of the module were developed with student centered learning as their 182 focus. Students are provided with background reading and assignments for out of class work 183 allowing them to connect the concepts and learning objectives of the module with previous 184 knowledge and skills from other courses. Each lesson period includes think-pair-share and other 185 student-centered activities. Students are encouraged to discuss their ideas with one another as 186 well as with the faculty involved, framed by a discussion of “biological literacy” as defined by 187 the Vision and Change Final Report (1), and focusing on how science is done in a real world 188 context, and how the practice of science has changed.

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189 The resources used in the various parts of the lesson are all freely available through the web and 190 students learn to access and assess these materials while creating their own knowledge and 191 understanding and have ownership of the hypotheses they develop, all important aspects of 192 inclusive teaching (47). Papers selected for class discussion are chosen to allow a discussion of 193 diversity of ideas and backgrounds of the authors and how diversity in its many forms 194 contributes to better problem solving (48). To further illustrate and model a scientific 195 community, the authors of this paper frequently sit in (virtually) on the hypothesis presentations 196 of the other authors’ students, leading to a robust discussion that enhances the students’ sense of 197 ownership in their project.

198

199 LESSON PLANS 200 We have implemented this overall lesson, using facets of the Hypothesis Development and 201 Proposal Module (Supplementary Material S2) that we have developed in a variety of settings, 202 ranging from a PUI Upper Level Capstone course to a Community College introductory lab 203 course (Course Descriptions and Syllabi are listed in Supplementary Material S3). While the 204 entire module is designed for faculty use we have created discrete parts of the module that are 205 suitable for pre-class reading by students – these are included in Supplementary Material S2. 206 Below are the various ways we have implemented the module in these settings.

207

208 PUI Upper level Writing Intensive Capstone Course

209 This course, a full semester CURE class, meets for two four hour lab periods each week for a 14 210 week semester. The overall lesson (module) is spread out over the first 6-7 weeks of the class 211 and uses the equivalent of 7 lab periods. During the intervening lab periods students are working 212 on CURE related wet lab experiments. The spacing of the components of the module allows 213 students to complete the various writing assignments associated with the module.

214 Lesson: Part 1: Project Background: uses 75 minutes of a lab Class Period-( powerpoint 1in 215 Supplementary Material S3-Section 1A)

216 Pre-Class preparation by students: Module Overview, part 1A

217

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218 In Class: As part of the introduction to the course, the philosophy of a CURE is outlined and the 219 whole semester previewed. The rationale and mechanism of Think Pair Share activities is 220 outlined, and the various writing requirements discussed including drafts , and revisions to give a 221 final version. Once a draft is turned in it is assessed using the grading rubric, returned to students 222 and they have a limited period of time (usually 4-5 days) to revise and resubmit for final grading. 223 They can earn back up to 50% of the points they missed on the draft version. Students are 224 randomly paired into groups of 2 which are retained for the whole semester ( if there are an odd 225 number of students, one group of three is created). We then proceed to the first part of the 226 course, Hypothesis Development and Proposal Writing. All powerpoints are posted on 227 Blackboard before the class except that the reveal slides for the Think, Pair, Share activities are 228 not included. After the class a version with the reveal slides and any other annotations to the 229 slides that occurred during the class are included.

230

231 Introduce the Specific Learning Goals of the module: powerpoint 1A slide 2

232 Discuss the rubrics for the three learning goals of the lesson.: powerpoint 1A slide 2-5

233 After the learning objectives of the module and the rubrics that will be used to assess student’s 234 progress are outlined- We have found it important to be explicit about the levels of performance 235 expected on the various assignments and explicit assessment rubrics shared with the students 236 help set the bar for performance- a series of Think Pair Share activities focusing on what Malate 237 Dehydrogenase is, what it does and where it is located in the cell orient students and set up the 238 first writing assignment of the module, The Introduction and background section.

239 First “Think, Pair, Share” slide 6

240 Draw out the Reaction Catalyzed

241 What is important about the reaction?

242 Reveal: slide 7

243 This activity sets the tone for the module and introduces the “Think like a chemist, Think like a 244 Biologist” theme used throughout the module. It also emphasizes the power of writing things out 245 and sets the scene for the MindMapping exercises that thread through the module/

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246 Second Think, Pair, Share – Slide 8

247 Add in what is known about enzyme structure-function

248 Reveal:– Slide 9

249 Third Think, Pair, Share – Slide 10

250 Roles in Metabolism

251 Reveal: - Slide 11

252

253 The first mini-lecture is to introduce concepts relevant to malate dehydrogenase structure and 254 function to prime the students for the literature searching/reading that is coming in the next part 255 of the overall lesson. We briefly talk about these topics as things they need to think about

256 Mini-Lecture: Introduce Location, Regulation, Interactions (metabolons) Slides 12-15

257

258 The second mini-lecture of the session introduces MindMapping which many of the students 259 have not come across in previous classes unless they have taken them with us!

260 Mini Lecture- what is a mind map? Slides 16-18

261

262 This mini-lecture is followed immediately with practice and in their lab group they do a TPS to 263 start a mindmap of the material covered in the class period

264 Fourth Think, Pair, Share

265 2 minute mindmap of today’s class

266 Homework: Finish MindMap individually

267

268 Lesson: Part 2: Uses a full lab class period: Finding and Reading the Literature: Why and 269 How?: Powerpoint 2 in appendix

270 Pre-Class preparation: Students read Enzymes: An Introduction, and Module Section 4A

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271

272 Part 2 starts with a mini quiz (ten minutes) that samples some of the main points of the 273 previous class in the module.

274 Mini Quiz-1

275

276 Mini-Lecture: What is a Good Hypothesis – Slide 3

277 This mini-lecture focuses on what makes a good hypothesis and highlights the appropriate 278 rationale for finding and reading the primary literature

279 Mini Lecture: Using the Primary Literature – Slides 4-8

280 How to find literature

281 Briefly reviews available sources of the primary scientific literature and emphasizes how to 282 search for literature using specific terms and author names and the fact that once you have found 283 a relevant paper it usually, in its introduction, leads you to many more useful papers!

284

285 In Class activity: How to read a paper: with class paper (choice of papers from provided list: 286 (Supplementary Material S6) Slides 9-21 (these follow the Literature Worksheet, Module 287 Section 5A)

288 Group TPS etc, in class build a mindmap of the paper

289 4 stages:

290 1. Title & Abstract 291 2. Introduction 292 3. Methods & Results 293 4. Discussion & Conclusions

294 Leading students through stepwise building a mindmap of the paper not only demystifies paper 295 reading but illustrates how little time it can take to get the major information from a paper.

296 Group Work & Homework: For the remainder of the class period students are asked to find 5- 297 10 papers that they find interesting and might be the foundation of the idea/hypothesis that they

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298 will start to develop over the next few class periods. As homework they individually MindMap 299 one paper and add to the MindMap of Background to Malate Dehydrogenase they started in the 300 previous class.

301 Students encouraged to reread part 1 of the module handout which contains an outline of the 302 major points about Malate Dehydrogenase to provide a context for their literature searching and 303 draft writing of their project background. They are expected to place the literature they find in 304 this context.

305 3-4 Days Later: Draft of Literature Background Due: (Grading Rubric in Supplementary 306 Material S7)

307 Students receive feedback on their draft of the literature background prior to next class period

308 Students review part 2 of the module handout prior to next class

309

310 Between part 2 and part 3 of the lesson, students use 2 lab periods and are learning how to use 311 the lab spectrophotometers and to determine basic kinetics of wildtype malate dehydrogenase

312

313 Lesson: Part 3: Uses a full Lab Period: Bioinformatics, Clustal see powerpoint 3

314 Pre-Class preparation: Students read Module Section 6A

315 Comments on Background Draft

316 Students have submitted their literature background drafts and I usually spend a few minutes 317 giving generic feedback on the drafts- highlighting common issues with drafts in general- they 318 receive individual copies of the scoring rubric used to assign a score for their draft. They get an 319 annotated copy of their draft: I usually annotate with questions for them to think about, or often, 320 queries about what they mean in a particular sentence, with comments such as “does this say 321 what you think it says?” or “is that what you mean?”

322

323 Mini_lecture: Developing Questions: Big Picture to Detail: build from Background to Malate 324 Dehydrogenase: Big Picture Unanswered questions. – Slides 4,5

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325

326 This mini-lecture focuses their thoughts on big picture issues related to protein structure 327 function relationships and malate dehydrogenase

328 Review What is a Good Hypothesis -Slide 6

329

330 Group TPS: Initial In Class Discussions for Hypothesis development: the Big Picture: Start a 331 MindMap- Slide 7

332 Student groups talk about potential areas of interest and generate initial ideas for their project

333

334 Mini lecture: What is Bioinformatics (and clustal omega or equivalent program) Slides 8-12

335 This minilecture gives a brief overview of what the bioinformatics that they are going to do is 336 based upon, and is followed by a brief think pair share activity (slide 13) where they decide upon 337 their big picture question and how they are going to construct their bioinformatics approach.

338

339 The majority of the Lab Period is used for the Bioinformatics Exercise-Based on the 340 Bioinformatics worksheet, Module Section 7A, and informed by their choices in the previous 341 TPS activity. Slides 14-22

342

343 In Class: group Mind Map development and presentation, Slides 23,24

344

345 At the end of the class I illustrate the bioinformatics activity with a sample clustal and discussion 346 of what conclusions were drawn from it.

347

348 This allows students to refine their thoughts and mindmap before the next class period.

349

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350 Lesson: Part 4 Uses a full Lab Period: PyMol (can easily be adapted to other molecular 351 visualization programs such as VMD, Jmol or Chimera) Powerpoint 4

352 Pre-Class preparation: Students read Module Section 8A, and Pre-Lab: students watch Pymol 353 tutorials.

354 In Class we start by revisiting What is a Good Hypothesis, (Slide 2) emphasizing the fact that it 355 must make testable predictions. This leads to the following discussion of non covalent 356 interactions in a mini lecture

357 Mini Lecture: Non Covalent Interactions and Enzyme Structure-Function: (Slides 3,4), which is 358 followed by an In class development of a mindmap of Non-covalent interactions and the role 359 they may play in enzyme structure and function which starts with a Think, Pair, Share activity 360 (Slide 5) and a reveal (slide 6)-

361 Depending upon the time available students work through the Molecular Visualization worksheet 362 (Module Section 9A)

363 Pymol Exercise: Students are asked to make pictures of residues they are interested in that show 364 what types of interactions they make – Slides 7,8, which ends with the challenge (slide 9) Start 365 Developing Hypothesis Detail- How do you think the residue(s) of interest interact with other 366 parts of the protein, the substrates etc

367 3-4 Days later: Revision of Literature Background Due: Supplementary Material: Rubric 368 1

369 Students revise initial draft based upon feedback and incorporate any additional background 370 resulting from their bioinformatics and molecular visualization work.

371

372 Lesson: Part 5 : Uses half a lab class Period: Predictions to Questions to Experimental 373 Approaches - powerpoint 5

374 Pre-Class preparation: Students read Module Section 10

375 Mini Quiz 2 on Assigned Reading (Supplementary Material S5-1A-2)

376

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377 Revisit “what makes a good hypothesis?” Slide 3

378 Recap – Slide 4

379 The purpose of this part of the lesson is to get students thinking about connecting their big 380 picture to specific, testable questions and by recapping briefly what they have already thought 381 about. This sets the tone for the rest of the class period.

382 Questions to Answer – Slides 5 & 6

383 Students seem to want to tell you what the mutant will do- the purpose of these slides is to 384 emphasize that what you are really trying to do is establish what the wildtype residue that you 385 are going to mutate does, and to connect what your predictions are to what experiments you can 386 perform. What experiments you want to perform revolves around asking the right questions and 387 then finding the technique that can help you answer the question. I usually give student groups 388 about 15 minutes to talk amongst themselves to generate their questions and encourage them to 389 discuss between lab groups as well as within lab groups- they are all making different hypotheses 390 and will probably end up having some common and some unique questions and benefit by the 391 cross fertilization of ideas and discussions. This mimics the process that would go on in a typical 392 research group meeting.

393

394 Mini-Lecture: Connecting Questions to Experiments Slides 7 & 8

395 The purpose of this mini lecture is to connect specific questions to experimental approaches that 396 they might consider proposing as part of their proposal. We don’t go into any detail- they have 397 seen some of these approaches described in their Biochemistry lecture course, some they have 398 not and will learn in detail as they proceed through the wet lab phase of the course. The goal is to 399 get them connecting questions to experimental approaches- they will get the detail later.

400 Group Work: Completing the MindMap - Slide 9

401

402 The remaining class time they focus on their Basic Hypothesis development and presentation 403 preparation. For the informal hypothesis presentation they are provided with a specific set of 404 guidelines. - Slide 10

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405

406 Between lesson part 5 and part 6 students first meet with faculty advisor to discuss planned 407 mutations, they design quikchange primers (which are ordered) and over 2 lap periods are 408 isolating plasmid DNA and performing quikchange to create their mutants,

409

410 Lesson: Part 6: Uses 3 Hours of a 4 hour Lab Period

411 Pre-Class preparation

412 Students read Part 3B of the Module Handout and review the Presentation Rubric 413 (Grading Rubric, Supplementary Material S8)

414 Presentations:

415 Each group presents their ideas and justification for their hypothesis, responds to questions from 416 faculty.

417 The class usually has 6-8 groups of students and the time allotted for discussion is adjusted to 418 make sure that there is about an hour left in class after the presentations.

419 Presentations and questions are followed by a Mini-Lecture on experimental approaches. 420 General class discussion about the types of experimental approaches that can be used, slides 4- 421 10, and a Think, Pair, Share activity to allow the students to further develop their ideas about 422 experiments to test their hypothesis.

423 If Appropriate: Develop ideas for potential Collaborations- what other types of data would help 424 them?

425 Their draft proposal, which are individual, are due usually 4-5 days later and are 426 anonymized and circulated for blind peer review using a peer review rubric which must 427 be completed and is due the day of peer review session

428

429 Between lesson parts 6 and 7 students use a lab period to prep their mutant DNA and send out 430 for sequencing

431

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432 Lesson: Part 7: Uses 2-3 Hours of a 4 hour lab period: Peer Review – powerpoint 7

433 Mini Quiz on Non Covalent Interactions using questions from LG2 Rubric- 434 (Supplementary Material S1)

435 Pre-Class preparation: Students review assigned proposals and fill in review rubric sheets: 436 (Supplementary Material S9- Peer Review Rubrics 1 & 2)

437 In Class

438 2-3 hours: “Study Section” Discussion and ranking of proposals: - write and turn in panel 439 summaries.

440 This session is run like an NSF Review Panel, where I play the role of the Program Officer. We 441 go through the proposals in an assigned order, each proposal has been reviewed by two students, 442 with a third student acting as a scribe for the discussion. Everyone in the class has access to the 443 proposal under discussion and follows the discussion. One of the assigned reviewers gives a 444 rating, a summary of the proposal and indicates strong and weak points. The second review adds 445 any additional points. The Program Officer then asks other students what they thought about 446 particular points raised by the reviewers. At the end of the discussion of a particular proposal the 447 Program Officer asks a student to indicate where the proposal should be ranked on a High, 448 Medium, Low scale. The session continues until all the proposals have been discussed and 449 categorized, often with questions from the program officer such as was this one as good as 450 proposal X that we discussed earlier. At the end of the session the group revisits the placement of 451 the proposals and finalizes the rankings. The scribes turn in their summary of the discussion of 452 the individual proposals and the session concludes with a brief discussion of what made certain 453 proposals stand out.

454 The next day students receive the various critiques from the study section together with my 455 graded rubric of their draft.

456 Lesson: Part 8: Uses a full lab period: Collaborations, Develop Further Experiments etc

457 I always leave one lab period where they can ask questions individually about their proposal and 458 the comments made while they are doing wet lab work. We usually have a Group discussion, 459 focused on developing further experiments based upon presentation discussion etc, When the

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460 class is collaborating with another class at a different university, this is also a chance to start to 461 develop specific collaborations as appropriate

462 The class usually meets on a Tuesday/Thursday and part 7 & 8 occur on Tuesday and Thursday 463 respectively. The students have til the end of the weekend (Sunday at 11.59pm) to turn in their 464 revised proposals which are then finally graded using the rubric for LG3.

465

466 Assessment of Learning Goals- see rubrics

467 LG 1: Proposal Introduction (Literature Review), Draft and Revision

468 LG 2: In Class Quiz on Non Covalent Interactions

469 LG 3: Proposal Presentation in class and final written proposal

470 Table 1A: Lesson Plan Timeline – Hypothesis Development and Proposal Module in a 471 Biochemistry Capstone Course

Activity Description Estimated Notes Time Project Introduction, Review of Protein Structure, Finding and Reading the Primary Literature 1. Give a big picture introduction of the research project Powerpoint 1 Part 1: 2. Give a general review of amino acid

structure, non-covalent interactions and Module Mini-lecture on 75 min their role in enzyme structure and handout Project function Introduction & Introduction 3. Introduction to Mind Mapping Background

Group exercise on dissecting i) Title and Part 2: Powerpoint 2 the Abstract ii) Introduction, iii) Results,

and iv) Discussion and Conclusions of Exercise on how to 240 min. an MDH paper read the Abstract lab period Module Create a stepwise mindmap of the paper. and Introduction of handout part Find, Read and Mindmap a paper on a primary literature 1A Malate Dehydrogenase Bioinformatics Tools to Develop a Hypothesis Part 3 Mini-lecture on Bioinformatics 30 min Powerpoint 3 approaches using amino acid sequences

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Mini-Lecture on bioinformatics 1. Use PubMed and Clustal w to search MDH sequences from various Module Class exercise organisms, and align selected sequences 210 min handout part using Clustal  to analyze enzyme active site, cofactor 1B binding site, etc.

Molecular Visualization to refine Hypothesis Review What is a good hypothesis 5 minutes Part 4 Mini-Lecture on Noncovalent interactions and their roles in protein 15 min Powerpoint 4 Mini-Lecture on structure and function

NonCovalent

Interactions Students make Pymol Pictures to Module illustrate their chosen amino acid residue 3.5 hours Handout part 2 Class Exercise and its potential interactions in MDH (to using PyMol other parts of MDH, to substrate, to cofactor etc)

From Hypothesis to Predictions and Experiments Revisit What is a good Hypothesis Part 5: 5 min

Recap roles of Non covalent interactions Developing 30 nin Powerpoint 5 in the context of what the selected predictions and residue does in the protein questions for the Module proposal handout part Defining Questions to ask 1-2 hours 3A Selecting appropriate Experimental

approaches

25 min Presentation of Hypothesis and Overview of Project Non-Covalent Interactions Quiz 30 min Part 6: Students present their hypothesis in 1-1.5 hours groups (2-3/group). Hypothesis includes Powerpoint 6 Presentation of background, amino acid sequence Hypothesis alignment, 3D structure of MDH active Module site and cofactor binding cite, and Handout 3B reasoning of their proposed mutation. As

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appropriate, a faculty from another institute joins in through video- Choosing conferencing to give students feedback. experimental approaches Mini-Lecture on Experimental 30min Approaches briefly reviews key aspects of available experimental approaches

Study Section: Peer Review & Critique of Proposals Students participate in a “panel review” Part 7 session of their draft proposals Summarize strong and weak points of all 3 hours Powerpoint 7 Peer Review draft proposals that are raised during discussion, Rank proposals Finalize Proposal Students revise their own proposals having received feedback from the panel Part 8 review, presentation questions and 3-4 hours faculty on their draft proposal. Open

time for students to ask follow up questions etc 472

473 TEACHING DISCUSSION 474 Four elements of the lesson stood out as being particularly successful for the students. 1). The 475 use of student constructed mind-maps at various stages of the lesson sequence fully engaged the 476 students and helped them connect both concepts and content during the module. 2). In our 477 experience students are not very good at reading papers and getting to key detail in the papers. 478 The exercise in part 2 of the lesson where the instructor models for the students how to break 479 down a paper and build a mind map from big picture to detail really helped students approach the 480 literature in a more engaged and constructive way and helps them with aspects of developing a 481 hypothesis and well as presenting information. 3)While students are good at memorizing 482 information about amino acid sidechains it is often disconnected from important concepts 483 necessary for deep understanding of the roles amino acid sidechains play in protein structure and 484 function. Visualizing side chains and connecting important properties to types of sidechains in 485 emphasized in the lesson, with a particular focus on non covalent interactions. We have found 486 that having a physical model of sidechains (such as those that can be obtained from 3D

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487 Molecular Designs) really helps students- ask students to sort the amino acid sidechain models 488 without giving them any other direction and then discuss what criteria they used to sort the 489 sidechains and they soon realize that there are many ways to categorize amino acid sidechains. 490 This is an important aspect of the 4th point, the use of molecular visualization as students start to 491 think in detail about their hypothesis and how structure function relationships play into their 492 proposal. It is also a great way to re-emphasize the role that Coulomb’s law plays in how 493 molecules look and behave. Revisit the equation describing Coulomb’s law and students quickly 494 realize that repulsions will play as important a role as attractions, and that local environment (the 495 local dielectric constant) can greatly influence sidechain and mainchain interactions. Also during 496 the exercise where they have to look at the potential interactions made in the molecule they 497 realize that both sidechain and mainchain interactions are important. It also helps students think 498 about dynamic structure as well as the static structure they see in a pymol image. The hypothesis 499 component of the module is developed as a lab group. While the presentations during the module 500 are joint, the final written proposal is individual. The goal of the presentations is to give feedback 501 to help with the final individual written proposal. (At the end of the semester each group 502 produces a presentation that they work on together but are “examined” individually and the 503 students are held individually responsible for answering questions about all aspects of the work.)

504

505 PUI Upper Level Biochemistry Laboratory Course

506 This course, a full semester CURE class, meets for one four hour lab period each week for a 13 507 week semester. The overall lesson (module) is spread out over the first 6 weeks of the semester. 508 Students enrolled in the class are junior and senior undergraduate majors in Biochemistry, 509 Chemistry, and Biology. 510 511 Lesson 1: Project background:

512 45 minutes: Discuss general concepts about enzymes, talk specifically about MDH: the reaction 513 it catalyzes and its role in the metabolism.

514 Students are introduced to the goal of this semester – ie developing and testing a hypothesis 515 about MDH structure/function.

516

517 At the end of the class period students are assigned HW#1 –( labeled JMOL HW#1_P2

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518 MDH coordinates_P3), a JMOL activity where the students are required to upload MDH 519 coordinates to JMOL and produce an image of MDH where key residues, the substrate mimic 520 Citrate and NADH are highlighted. 521

522 Lesson 2: Finding and reading literature:

523 20 minutes: Brief lecture describing how to use and find Primary literature

524

525 180 minutes: In class activity – students are provided with a MDH paper one section at a time 526 starting with the Abstract. Working in pairs the students read each section and answer a 527 provided questions that correspond to the respective section and then we report back as a class. 528 After one section is completed we move onto the next section until the paper is completed.

529

530 Lesson 3: Bioinformatics

531 20 minute: Brief lecture describing the structure of MDH and the reaction mechanism of MDH.

532

533 100 minute: Working in pairs students complete an in class worksheet where they are guided 534 through a series of activities to help them develop their hypothesis (Bioinformatics 535 Worksheet_P5). Students upload coordinates of wgMDH (MDH coordinates_P3) to Jmol again, 536 this time they start measuring distances between residues in the active site and the substrate. 537 Students then use Uniprot to look for amino acids that are conserved across multiple species.

538

539 At the end of the class period students are assigned Hypothesis Development Homework, 540 HW#2_P6 – students must submit hypothesis as well as a list of relevant MDH literature that 541 they are going to use for their proposal.

542

543 Lesson 4 – students pairs have individual meetings with Professor to discuss their current 544 hypothesis.

545

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546 Lesson 5 (2 lab periods after lesson #4) –

547 Student pairs turn in their written proposals and give presentations to the class (and outside 548 collaborators) describing their hypothesis, the rationale behind it, and how they are going to test 549 it.

550 Assessment of Learning Goals

551 LG 1: HW #1 and HW#2

552 LG 2: In class open notebook Quiz

553 LG 3: Written proposal and presentation

554 Table 1B: Lesson Plan Timeline – MDH Bioinformatics and Hypothesis for mCURE at a PUI

Activity Description Estimated Notes Time Project Introduction and MDH background 1. Give a big picture introduction of the Mini-lecture Lecture slides in research project on Project and 30 min the Supporting 2. Talk about MDH the reaction in MDH File P1 catalyzes and its role in metabolism JMOL activity where students are required to upload MDH coordinates to Supporting File HW #1 JMOL and produce an image of MDH 60-90 min P2 highlighting key residues, the substrate mimic Citrate and co-factor NADH Using Primary literature Mini-Lecture How to use PUBMED, ScienceDirect, on Primary 20 min Google Scholar literature Students are provided with a MDH paper one section at a time starting with the Guided Abstract. Working in pairs the students Guided MDH manuscript read each section and answer a provided manuscript 3 hours worksheet in the questions and then we report back as a review supporting file, class. After one section is completed we P4. move onto the next section until we cover the entire paper.. Bioinformatics and Hypothesis Development

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Working in pairs students complete an in class worksheet where they are guided through a series of activities to help them develop their hypothesis. Students P5-Hypothesis In class upload coordinates of wgMDH to Jmol Development 2 hours worksheet again, this time they start measuring P3-MDH distances between residues in the active coordinates site and the substrate. Students then use Uniprot to look for amino acids that are conserved across multiple species. Students must submit hypothesis as well HW#2 as a list of relevant MDH literature that >3hrs P6 they are going to use for their proposal. Written proposal and presentation Student pairs turn in their written Written proposals and give presentations to the proposal and class (and outside collaborators) 15-20 min

oral describing their hypothesis, the rationale presentations presentation behind it, and how they are going to test it. and the substrate.

555 TEACHING DISCUSSION 556 The goal of this module is to help guide students to develop a testable hypothesis about Malate 557 Dehydrogenase. The students taking this course often times have only a superficial 558 understanding of Malate Dehydrogenase. To overcome this, as simple as it may sound, it is 559 necessary to talk about the varying aspects of Malate dehydrogenase (structure, reaction 560 mechanism, substrate binding, etc) every single class. It is important that the students feel as 561 though they are becoming experts on MDH. The tools that are most helpful for the students in 562 identifying their amino acid to mutate are Jmol (lesson 1 and 3) and Uniprot (lesson 3). The 563 visualization of the structure of MDH and the specific roles amino acid side chains play in its 564 functionality in conjunction with the evolutionary conservation of these amino acids is critical to 565 the student’s ability to formulate their hypothesis. Students at this stage in their career (Junior or 566 Senior) find reading primary literature difficult and often struggle extracting the key elements 567 from the paper. In Lesson 2 students are guided through a MDH paper by the instructor. This 568 exercise takes time, but is really essential to the student learning as it continues to build their 569 knowledge of MDH and helps develop a skill that they will need to write their proposals. 570 Students have very little experience formulating a testable hypothesis. Initial hypotheses often

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571 take the form of “Change this Amino Acid and observe what happens”. HW#2 provides the 572 students with guidelines to writing a hypothesis, but more importantly in provides the instructor 573 with an assessment of where the students are in their hypothesis development. The instructor 574 meetings with each group (Lesson 4) to discuss their hypothesis and virtual meetings with 575 outside instructors/students from outside institutions (Lesson 5) typically lead to hypotheses that 576 are significantly different from their initial draft.

577

578 Community College

579 LESSON PLAN FOR PARTIAL COURSE CURE IN AN INTRODUCTORY COURSE 580 WITH FEW OR NO PREREQUISITES 581 LESSON PLAN FOR PARTIAL COURSE CURE IN AN INTRODUCTORY COURSE 582 WITH FEW OR NO PREREQUISITES 583

584 Prior to the lessons: These lessons are introduced approximately 5 weeks into the semester. 585 Students have already been introduced to biological molecules such as proteins, cell structures, 586 and the scientific process.

587

588 Lesson Part 1: Project Introduction and Bioinformatics: 170 minute Lab Period

589 Mini Lecture: 30 min. Students are introduced to enzymes as catalysts for reactions. Malate 590 dehydrogenase’s reactions are used as examples. Enzymes are proteins; students are reminded of 591 a prior lesson about levels of protein organization; structures of amino acids. Enzymes illustrate 592 the common theme of a relationship between structure and function. Students are told that they 593 will be investigating the structure of MDH, and considering the effects of changes to its 594 structure. We can find out what is known about MDH and its structure through protein 595 databases and software.

596 In-class guided hands-on activity with laptops: 140 min. Introduction to PDB and Pymol: 597 Introduction to biological databases and software to visualize structures, beginning with a short 598 video about PDB. Students get laptops, and follow along as instructor uses PDB for a few 599 simple operations. Next, instructor projects the first of six videos about the use of PyMol, while 600 pausing, demonstrating on instructor’s computer, with screen projected, and students follow

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601 along, trying the tasks on their laptops. Students are given the assignment which leads them 602 through the use of PDB and PyMol to investigate malate dehydrogenase. A limited number of 603 specific MDH mutations are included in the assignment for students to visualize while using 604 PyMol. Students work in teams of 2-3, each with a computer. Instructor circulates, gives 605 simple help, encourages students to watch the PyMol instructional videos as they progress 606 through the tasks in the assignment. After this time in class, most students are comfortable 607 enough to continue work outside of class.

608 Homework: Students complete remainder of assignment. Assignment is due in one week.

609 Reference Handout: Diagram showing amino acid structures, names, and abbreviations.

610 Resources: videos about PDB, PyMol., links provided on word doc.

611 Assignment: MDH simplified Bioinformatics and PyMol

612 Lesson Part 2: Reading Scientific Papers and Hypothesis Development: 170 minute Lab 613 Period

614 Mini Lecture: 20 min. Explanation of types of scientific literature, difference between primary 615 literature and review papers. PubMed and Google Scholar searches are demonstrated. Students 616 are given a printed paper, Cox et al, 2005. Instructor guides students to look at the structure of 617 the sections of the paper.

618 Student team worktime:2 hours and 30 min. Assignment is given, to be started in class. Each 619 student answers the questions in his/her own words, but they are encouraged to help each other 620 with the dissection of the paper, and discuss as a team. The assignment guides students through 621 the main sections of the paper, but with a focus on finding some key information related to the 622 basic biological context of malate dehydrogenase, and the roles of specific regions where the 623 mutations (in the PyMol assignment) are. Students also will select another paper to read as 624 relates to one particular mutation (see below).

625 Student selection of focus for hypothesis: Before the end of the lab, while students are still 626 working on the assignment, students are told that each team will be able to focus on just one of 627 the limited number of mutations they modeled in their PyMol exercise. Each team makes a 628 ballot ranking their choices. Instructor uses the ballots to assign mutations to each team. 629 Depending on team size and total team number, multiple teams may work with the same

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630 mutation. In a course without teaching assistants, most instructors find that three mutations is a 631 manageable number; attempting to work with more than three may provide undue strain on 632 materials, instructor attention, and other scarce resources.

633 Homework: Students complete the remainder of the assignment, including formulating a 634 hypothesis about “their team’s” particular mutation, due in one week.

635 Assignment: MDH lit review and hypothesis.

636 Assessment of Learning Goals

637 LG 1: MDH simplified Bioinformatics and PyMol, Summary of Enzyme Results, Poster

638 LG 2: Calcuations of Specific Activity and Turnover, Summary of Enzyme Results, Poster.

639 LG 3: MDH Lit Review and Hypothesis, Poster.

640 Table 1C:

641 Lesson Plan Timeline – MDH Bioinformatics and Hypothesis for mCURE at a Community 642 College

643

Activity Description Estimated Notes Time Introduction to Enzymes, Protein Databases, and Software for Visualization of Protein Structure 1. Enzymes are protein catalysts 2. Malate Dehydrogenase, Mini-lecture: role in cells. Diagrams drawn on whiteboard, introduction to 3. Levels of protein students referred to figures in textbook, enzymes and organization; amino 30 min a handout showing amino acid Malate acids. structures and properties of R groups. Dehydrogenase 4. Relationship of structure and function. 5. Databases and software. Access and 1. Students use PDB website 20 min Explore laptops, find PDB https://www.rcsb.org/

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Bioinformatics in a browser while tools watching a video 8-9 min video intro to PDB about PDB. https://pdb101.rcsb.org/ 2. Download MDH structure. 4-5 min video intro to PyMol 3. Watch video https://youtu.be/Bhqm7JrvDBY intro to PyMol and download free Open Source Free PyMol (new version location) https://github.com/schrodinger/pymol- open-source

Students work in Basic Rendering Video teams of 2-3 on an https://youtu.be/arMljkKcu4E assignment, use Finding Important Amino Acids PDB and PyMol to https://youtu.be/KHsIdS5-kKQ investigate malate Measurements and Mutagenesis dehydrogenase and https://youtu.be/xLSPYgyB8J4 a limited number of Hydrophobics and Structure Overlay Guided specific MDH https://youtu.be/4UOBjbPp9MM Bioinformatics mutations. 2 hours How to make a Publication Image Assignment Instructor https://youtu.be/88qvsktfH0c circulates, helps, How to make a movie encourages use of https://vimeo.com/44801178 instructional videos. Work unfinished Bioinformatics Assignment: Protein during class time is Structure provided in Supporting Files. completed at home, due in one week. Using the Literature, along with prior Bioinformatics work to Develop a Hypothesis Brief lecture on use of PubMed and Google Scholar, Introduction to review papers and scientific 20 min primary literature, literature overview of the structure of a scientific paper. Students review one Class worktime common paper in 2 hours Literature Review and MDH to review a teams. Teams will and 30 Hypothesis Formation assignment is particular later chose an minuts provided in Supporting Files. paper additional paper as

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a second reference. Assignment has guiding questions about sections of the paper, but with a focus on basic biological context of malate dehydrogenase, roles of specific regions of mutations investigated in the PyMol assignment. Before the end of the lab, each team 10 min ranks choice of Student (sometime mutations for Selection of within the hypothesis focus. Focus for 2 hour and Instructor assigns Hypothesis 30 min mutations to each above) team.

Student teams choose second paper based on “their” mutation. Final question of the assignment prompts hypothesis Hypothesis development,

Development connecting information from the literature review and the PyMol exercise from the prior week. Assignment is due in two weeks. 644

645 TEACHING DISCUSSION

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646 We have used a short version of this module in an introductory biology course at a community 647 college. The introductory biology course is often taken concurrently with the introductory 648 chemistry course, and neither courses have pre-requisites beyond college level math and reading 649 readiness. At this open-access institution, we cannot assume prior chemistry or biology 650 knowledge, so the introduction to this project includes lessons about proteins, levels of structure, 651 and their monomers, amino acids. This module is used at the same time as enzymes are being 652 introduced in the lecture. Typically, the flow of genetic information is covered as one of the last 653 topics of the semester, so it has not been introduced before this module is used. Therefore the 654 connection of DNA sequence to protein sequence and structure in living organisms is not yet 655 clear to the students, though when that topic is introduced later, this malate dehydrogenase work 656 can be referred to as an illustrative example.

657 The assignments and exercises given have been written to combine learning about the resources 658 while using them to learn about malate dehydrogenase. Thus, students learn about the existence 659 and use of PDB, PyMol, PubMed, while using them to answer specific questions about malate 660 dehydrogenase. While some students at this level are able to work with a very open-ended set of 661 questions, many others lack confidence and prior experience with that level of challenge and 662 unfortunately internalize typical discomfort with the unknown into “evidence” that they are not 663 capable of becoming scientists. To make the exercise achievable by most students, we restrict 664 the focus of the hypothesis development toward very specific suggestions. The assignments are 665 also started in-class with instructor guidance, and students work in teams, so that student comfort 666 level is increased. It’s been helpful to acknowledge and discuss how it feels to not-know, that 667 the instructor feels this way sometimes too, and work to practice reacting with curiosity rather 668 than anxiousness.

669 The assignments were designed to isolate and encourage the use of evidence and concentrate on 670 ideas, rather than writing. Scientific communication is introduced, as the students are asked to 671 note the structure of the papers they read, but they are not asked to write a stand-alone proposal 672 at this point. Later in the semester, they will put several separately generated pieces of their 673 writing together in a presentation of their results in the form of a scientific poster.

674 In this shortened version of the CURE, students were taught to use the protein data base and the 675 protein visualization software, and used these with published literature in forming their

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676 hypothesis; an alignment and evolutionary analysis were not included. The section of the 677 module used directly was the general introduction to malate dehydrogenase. The “MDH 678 Simplified Bioinformatics PDB and PyMol” assignment was based on Part 2: Exploring 679 Structure-Function Relationships in Malate Dehydrogenase II: Using Molecular Visualization 680 tools. The “MDH Lit Review and Hypothesis” is a much condensed version of Part 1A (primary 681 literature) and Part 3A (making predictions).

682

683 4 yr Research 1 University

684 LESSON PLAN 685 The Lesson plan was used in an mCURE biochemistry lab course. Students enrolled were 686 junior and senior undergraduate majors in Biochemistry, Chemistry, and Biology, at an R1 687 university. 688 689 Lesson Part 1: Project introduction and Review of protein structure - 50 minute Lecture 690 Period 691 Introduction of MDH project. Lecture Slides_R1, slides 2,3 692 Mini lecture part 1: a general review of amino acid structure, non-covalent interactions, and the 693 role they may play in enzyme structure and function. Lecture Slides_R1, slides 4,5 694 Mini lecture part 2: How to find primary literature and How to read a paper. Lecture Slides_R1, 695 slides 6-8 696 In Class activity: Group exercise on How to read Abstract and Introduction. 697 Assignment: students work in pairs, and write a draft of MDH introduction using at least two 698 papers. 699 700 Lesson Part 2: Bioinformatics – 30 minute Lecture Period plus 3 Hour Lab Period 701 Mini Lecture: Introduction of Bioinformatics. Lecture Slides_R1, slides 9-13 702 Lab Period: Students work in pairs to exercise bioinformatic tools on their laptops. For the first 703 part, students were directed to www.uniprot.org and search for MDH sequences, and then use at 704 least 5 sequences to perform alignment. For the second part, students watch Chimera video 705 tutorials and identify residues and hydrogen bonds in and around MDH active site. A list of 706 MDH mutants was provided for students to visualize in the 3D structure.

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707 Assignment: Students work in pairs to propose their hypothesis from the provided mutant list. At 708 the beginning of next lecture period students review another pair’s hypothesis and give feedback. 709 710 Lesson Part 3: Presentation of Hypothesis - 3 hour Lab Period. Lecture Slides_R1, slides 14 711 Each group gives a 10 minute presentation on their hypothesis, including a brief background, 712 results of sequence alignment with annotation, 3D structure image of mutation site, and 713 hypothesis of its effect on MDH function. In a collaborative mCURE, a faculty from another 714 institute joins in through video-conferencing and gives students feedback. 715 Assignment: Students incorporate feedback to revise their hypothesis 716 717 Assessment of Learning Goals 718 LG1: MDH Literature Review and Non-Covalent Interactions 719 LG2: In-class Bioinformatics Worksheet and Hypothesis Draft 720 LG3: Hypothesis Presentation in class and Hypothesis Revision

721

722 Table 1D: Lesson Plan Timeline – MDH Bioinformatics and Hypothesis for mCURE at an R1 723 University

Activity Description Estimated Notes Time Project Introduction and Review of Protein Structure 1. Give a big picture introduction of the research project Mini-lecture on 2. Give a general review of Project amino acid structure, non- Lecture Slides_R1, slides Introduction and covalent interactions and their 25 min 1-6 finding primary role in enzyme structure and literature function 3. Show how to use PubMed to search for primary literature Exercise on how to read the Group exercise on dissecting Lecture Slides_R1, slides Abstract and the Abstract and Introduction 25 min 7,8 Introduction of a of an MDH paper primary literature

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Students work in pairs, and write a draft of MDH Assignment introduction using at least two papers Bioinformatics Tools to Develop a Hypothesis Mini-Lecture on common Bioinformatics Introduction on PDB, Lecture Slides_R1, slides 9- 30 min tools for UniProt, Chimera 13 structural biochemistry 1. Use UniProt to search MDH sequences from various organisms, and align selected In-class exercise sequences to analyze enzyme on using common Bioinformatics_Hypothesis active site, cofactor binding 2 hours Bioinformatics Worksheet_R1 site, etc. tools 2. Watch Chimera tutorial videos. 3. Bioinformatics Worksheet Presentation of Hypothesis Students present their hypothesis in groups (2- 3/group). Hypothesis includes background, amino acid sequence alignment, 3D structure of MDH active site Presentation of Lecture Slides_R1, slides and cofactor binding cite, and 3 hours Hypothesis 14 reasoning of their proposed mutation. In a collaborative mCURE, a faculty from another institute joins in through video-conferencing to give students feedback. 724

725 TEACHING DISCUSSION

726 The biggest challenge in a mCURE is to choose key research elements that are sufficient for 727 students to develop a meaningful hypothesis within a relatively short period of time frame about 728 3 lab periods.

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729

730 In lesson part 1, the goal is to guide students to use primary literature to have an idea about the 731 enzyme malate dehydrogenase and its significance. Reading primary literature is challenging, so 732 the focus is placed on the ‘Abstract’ and ‘Introduction’ sections. ‘Abstract’ is short enough for 733 in-class exercise guided by the instructor to give students a big picture of the paper. 734 ‘Introduction’ is relatively easy to understand and can provide general background about MDH. 735 After the assignment on writing a draft of MDH introduction using at least two papers, students 736 can have a general idea about the enzyme. A review on amino acid structure and non-covalent 737 interactions is given to prime students for the next lesson on bioinformatics.

738

739 In lesson part 2, the goal is to guide students to use bioinformatics tools to explore the effect of 740 amino acid mutation on non-covalent interactions, hence the kinetics of the enzyme. Students are 741 often found lost if they are given the whole protein sequence to hypothesize from. An alternative 742 strategy is to give them a small set of mutants to choose from, and hypothesize the effect of the 743 mutation on enzyme function. Instructor first introduces PDB, UniProt, and Chimera, and then 744 guides students to work through a bioinformatics worksheet, which includes sequence alignment 745 of MDH using UniProt and 3D visualization of active site, cofactor binding site, and mutation 746 site using Chimera.

747

748 Now with a general knowledge of MDH and visualization on the enzyme structure as well as the 749 mutation site, students can use their knowledge of the role of amino acid side chains on non- 750 covalent bonding to put together an initial hypothesis on the effect of mutation on enzyme 751 function. During lesson part 3 the presentation of hypothesis, students get feedback from the 752 instructor or faculty from collaborating institution and modify their hypothesis. This is usually a 753 very valuable learning experience for the students.

754 CONSENSUS TEACHING DISCUSSION 755 The lesson plans presented here were developed as part of the Malate Dehydrogenase CUREs 756 Community (MCC). MCC CUREs were instituted with two goals, 1) to provide high impact 757 teaching practices for students, and 2) to create new knowledge on structure function

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758 relationships in Malate Dehydrogenase that can be submitted for publication in peer-reviewed 759 journals.

760

761 While all MCC CUREs involve students in Scientific Background, Hypothesis Development, 762 Proposal, Experiments/Teamwork to test hypothesis, Data Analysis and Conclusions, and 763 Presentation, one of the distinguishing features of MCC CUREs is the emphasis on student 764 generated hypotheses.

765 All student generated hypotheses should contain the following elements:

766 Big Picture- why is this important to science, to society 767 768 Background Information 769 Preliminary Experiments/Observations, What is known/published 770 771 Specific Hypothesis 772 773 Predictions arising from the hypothesis 774 775 Tests of the predictions 776 What types of information do you need to falsify/support the hypothesis 777 778 And, as outlined in figure 1 should culminate in some type of presentation of the hypothesis and 779 proposed research.

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781 As MCC resources grow, we have available a number of mutants that have validated sequences, 782 and in many cases pre-existing data that has been validated by subsequent experiments after the 783 culmination of the class These mutants and their validated data can be made available for use in 784 MCC CURE Classes.

785 We propose that this information be available to students as they develop their own hypotheses 786 in an MCC CURE Class. For example, they will be able to see what mutants have been made 787 previously and any validated data on those mutants. The data base of this information does NOT 788 contain any information as to what hypotheses were developed with respect to the mutation, nor 789 are any suggestions as to what hypotheses were being tested be given to students.

790 There are several ways this information can be used in a class.

791 1. Students could be given a selection of these mutants with the validated background 792 information and asked to develop a hypothesis as to what function they think the wildtype 793 residue played in the protein, and what they think the mutation will do to the structure- 794 function relationships of the enzyme and devise and conduct experiments to explore their 795 hypothesis. 796 2. Students could be given a “big picture” question. For example, what governs folding of 797 Malate Dehydrogenase? As part of the accessible background information about MDH, 798 the list of validated mutants and associated effects would be provided to the students. 799 They would use this information in much the same way as they would use published 800 information and would develop their own hypothesis about (in this example that may or 801 may not make use of one of the validated mutations. At the instructor’s discretion, they 802 could be restricted to using one of the pre-existing mutations. 803

804

805 Each of us has used pre- and post assessment using EDAT (Experimental Design Ability Test) 806 (49) in our courses which specifically targets student’s ability in designing experiments- a key 807 component of the lesson. EDAT scores across the MDH CUREs Community showed a 808 significant increase in CURE courses from pre to post test and that the increase was significantly 809 higher (at p=0.001) than in control non-CURE courses.

810

811 Finally, one aspect of the lesson that we have all found extremely effective is the impact of 812 sitting in on one-another’s student presentations of their hypothesis. We have used Skype or

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813 Zoom to virtually sit in on presentations and students are energized by having someone other 814 than their own instructor involved. This aspect has in some cases led to student collaborations 815 between institutions. In both, it seems clear that student ownership of their project is enhanced 816 by these interactions. Through the Malate Dehydrogenase CUREs Community we are in the 817 process of assessing this aspect in detail as part of our current IUSE grant.(50)

818 819 References 820 1. Vision and Change in Undergraduate Biology Education: A View for the 821 21st Century,. 2009, AAAS. 822 2. Benvenuto, M. Educational reform: why the academy doesn’t change. Thought and Action. 823 2002,18, 63–74 824 3. Bell, E., The future of education in the molecular life sciences. Nat Rev Mol Cell Biol, 2001. 825 2(3): p. 221-5. 826 4. in Bio2010: Transforming Undergraduate Education for Future Research Biologists. 2003: 827 Washington (DC). 828 5. Wood, W.B., Inquiry-based undergraduate teaching in the life sciences at large research 829 universities: a perspective on the Boyer Commission Report. Cell Biol Educ, 2003. 2(2): p. 112-6

830 6. Graham, M.J., et al., Science education. Increasing persistence of college students in STEM. 831 Science, 2013. 341(6153): p. 1455-6.

832 7. Russell, S.H., M.P. Hancock, and J. McCullough, The pipeline. Benefits of undergraduate 833 research experiences. Science, 2007. 316(5824): p. 548-9. 834 8. Seymour. E., H.A.B., Laursen. S.L., Deantoni. T., Establishing the benefits of research 835 experiences for undergraduates in the sciences: first findings from a three-year study. Sci Educ, 836 2004. 88: p. 493-534. 837 9. Eagan, M.K., Jr., et al., Making a Difference in Science Education: The Impact of 838 Undergraduate Research Programs. Am Educ Res J, 2013. 50(4): p. 683-713. 839 10. Crowe, M.a.B.D., CUR Quart, 2008. 28: p. 43-50. 840 11. Lopatto, D., Undergraduate research as a catalyst for liberal learning. Peer Rev, 2006. 8: p. 841 22-25.

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842 12. Lopatto, D., Science in Solution: The Impact of Undergraduate Research on Student 843 Learning. Research Corp, 2009. 844 13. Luckie, D.B., et al., Infusion of collaborative inquiry throughout a biology curriculum 845 increases student learning: a four-year study of "Teams and Streams". Adv Physiol Educ, 2004. 846 28(1-4): p.199-209. 847 14. Myers, M.J. and A.B. Burgess, Inquiry-based laboratory course improves students' ability to 848 design experiments and interpret data. Adv Physiol Educ, 2003. 27(1-4): p. 26-33. 849 15. Rissing, S.W. and J.G. Cogan, Can an inquiry approach improve college student learning in 850 a teaching laboratory? CBE Life Sci Educ, 2009. 8(1): p. 55-61. 851 16. Hensel, N., Characteristics of Excellence in Undergraduate Research (COEUR). 2012, 852 Washington DC: Council on Undergraduate Research. 72.

853 17. Boyer Commission on Educating Undergraduates in the Research University. Reinventing 854 Undergraduate Education: A Blueprint for America's Research Universities. State University of 855 New York at Stony Brook for the Carnegie Foundation for the Advancement of Teaching: Stony 856 Brook, NY, 1998.

857 18. Lopatto, D., et al., A central support system can facilitate implementation and sustainability 858 of a Classroom-based Undergraduate Research Experience (CURE) in Genomics. CBE Life Sci 859 Educ, 2014. 13(4): p. 711-23. 860 19. Auchincloss L. C.; Laursen S. L.; Branchaw J. L.; Eagan K.; Graham M.; Hanauer D. I.; 861 Lawrie G.; McLinn C. M.; Pelaez N.; Rowland S.; Towns M.; Trautmann N. M.; Varma-Nelson 862 P.; Weston T. J.; Dolan E. L. Assessment of course-based undergraduate research experiences: a 863 meeting report. CBE—Life Sci. Educ. 2014, 13, 29–40. 864 20. Corwin, L.A., M.J. Graham, and E.L. Dolan, Modeling course-based undergraduate 865 research experiences: an agenda for future research and evaluation. CBE Life Sci Educ, 2015. 866 14(1): p. es1. 867 21. Denofrio, L.A., et al., Mentoring. Linking student interests to science curricula. Science, 868 2007. 318(5858): p. 1872-3. 869 22. Lopatto, D., Undergraduate research experiences support science career decisions and 870 active learning. CBE Life Sci Educ, 2007. 6(4): p. 297-306. 871 23. Dolan, E.L. and J.P. Collins, We must teach more effectively: here are four ways to get 872 started. Mol Biol Cell, 2015. 26(12): p. 2151-5.

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873 24. Lopatto, D., Survey of Undergraduate Research Experiences (SURE): first findings. Cell 874 Biol Educ, 2004. 3(4): p. 270-7. 875 25. Kloser, M.J., et al., Integrating teaching and research in undergraduate biology laboratory 876 education. PLoS Biol, 2011. 9(11): p. e1001174.

877 26. Lopatto, D., et al., Undergraduate research. Genomics Education Partnership. Science, 878 2008. 322(5902): p. 684-5. 879 27. Lopatto, D. CURE Survey. Jan 10, 2016]; Available from: 880 http://www.grinnell.edu/academics/areas/psychology/assessments/cure-survey. 881 28. D, L., The essential features of undergraduate research. CUR Quart, 2003. 24: p. 139-142. 882 29. Hanauer, D.I. and E.L. Dolan, The project ownership survey: measuring differences in 883 scientific inquiry experiences. CBE Life Sci Educ, 2014. 13(1): p. 149-58. 884 30. Rahm. J., M.H.C., Hartley. L., and Moore J.C., The vlue of an emergent notion of 885 authenticity: examples from two student/teacher scientist partnership programs. J Res Sci Teach, 886 2003. 40: p. 737-756. 887 31. Bell, E., Using research to teach an "introduction to biological thinking". Biochem Mol Biol 888 Educ, 2011. 39(1): p. 10-6.

889 32. Bell J. K.; Eckdahl T. T.; Hecht D. A.; Killion P. J.; Latzer J.; Mans T. L.; Provost J. J.; 890 Rakus J. F.; Siebrasse E. A.; Bell J. E. CUREs in biochemistry-where we are and where we 891 should go. Biochem Mol Biol Educ. 2016, 45, 7–12.

892 33. The Malate Dehydrogenase Cures Community. 893 https://mdh-cures-community.squarespace.com (accessed August 15, 2019).

894 34. Tansey, J. T.; Baird, T. Jr.; Cox, M. M; Fox, K. M.; Knight, J.; Sears, D.; Bell, E. 895 Foundational concepts and underlying theories for majors in biochemistry and molecular 896 biology. Biochem. Mol. Biol. Educ. 2013, 41, 289–296. 897 898 35. White, H. B.; Benore, M. A.; Sumter, T. F.; Caldwell, B. D.; Bell, E. What skills should 899 students of undergraduate biochemistry and molecular biology programs have upon graduation? 900 Biochem. Mol. Biol. Educ. 2013, 41, 297–301. 901 902 36. Wright, A.; Provost, J.; Roecklein-Canfield, J. A.; Bell, E. Essential concepts and underlying 903 theories from physics, chemistry, and mathematics for “biochemistry and molecular biology” 904 majors. Biochem. Mol. Biol. Educ. 2013, 41, 302–308. 905

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906 37. CourseSource Biochemistry and Molecular Biology Learning Framework. 907 https://www.coursesource.org/courses/biochemistry-and-molecular-biology

908 38. Yannis Hadzigeorgiou, Persa Fokialis1, Mary Kabouropoulou, “Thinking about Creativity in 909 Science Education”., Creative Education 2012. Vol.3, No.5, 603-611 Published Online 910 September 2012

911 39. Paul K. Strode, “Hypothesis Generation in Biology: A Science Teaching Challenge & 912 Potential Solution”., The American Biology Teacher, 77(7):500-506.

913 40. Teacher Guide: “Using Science News to Develop a Research Question and Hypothesis”

914 SN Educator Guide, April 29, 2017, Research in Brierf

915 41. Stefan Mark Irby, Nancy J. Pelaez, and Trevor R. Anderson 916 “How to Identify the Research Abilities That Instructors Anticipate Students Will Develop in a 917 Biochemistry Course-Based Undergraduate Research Experience (CURE)”, CBE—Life Sciences 918 Education • 17:es4, 1–14, Summer 2018 919 42. Cissy J. Ballen Carl Wieman, Shima Salehi, Jeremy B. Searle, and Kelly R. Zamudio 920 “Enhancing Diversity in Undergraduate Science: Self-Efficacy Drives Performance Gains with 921 Active Learning”. CBE—Life Sciences Education • 16:ar56, 1–6, Winter 2017 922 43. Joshua Premo, Andy Cavagnetto, and William B. Davis “Promoting Collaborative 923 Classrooms: The Impacts of Interdependent Cooperative Learning on Undergraduate Interactions 924 and Achievement”, CBE—Life Sciences Education • 17:ar32, 1–16, Summer 2018 925 44. Elisa M. Stone “Guiding Students to Develop an Understanding of Scientific Inquiry: A 926 Science Skills Approach to Instruction and Assessment”, CBE—Life Sciences Education, Vol. 927 13, 90–101, Spring 2014

928 47. Jennifer Loertscher, Jennifer E. Lewis, Allison M. Mercer and Vicky Minderhout, 929 “Development and use of a construct map framework to support teaching and assessment of 930 noncovalent interactions in a biochemical context” , Chem. Educ. Res. Pract., 2018, 19, 1151— 931 1165 932 47. Kimberly D. Tanner, “Structure Matters: Twenty-One Teaching Strategies to Promote 933 Student Engagement and Cultivate Classroom Equity”., CBE Life Sci Educ. 2013 Fall; 12(3): 934 322–331

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935 48. Katherine W. Phillips , “How Diversity Makes Us Smarter” - Scientific American. 936 https://www.scientificamerican.com/article/how-diversity-makes-us-smarter/ 937 October 1, 2014 —

938 49. Sirum K.,, The experimental design ability test (EDAT). Bioscience: Journal of College 939 Biology Teaching, 2011. 37(1): p. 8-16. 940 50. NSF-1726932 EHR-IUSE “MDH CUREs Community, a Protein Centered Approach to 941 CUREs”, $598,666, 9/1/2017 to 8/20/2020 942 ACKNOWLEDGMENTS 943 The materials presented here have in large part been developed as part of an NSF funded IUSE 944 grant: NSF-1726932 EHR-IUSE “MDH CUREs Community, a Protein Centered Approach to 945 CUREs”, $598,666, 9/1/2017 to 8/20/2020, Principal Investigator Ellis Bell, Co Investigators 946 Jessica Bell and Joseph Provost. 947

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948 Table and Figure Legends

949 TABLES

950 Table 1A: Lesson Plan Timeline – Hypothesis Development and Proposal Module in a 951 Biochemistry Capstone Course

952 Table 1B: Lesson Plan Timeline – MDH Bioinformatics and Hypothesis for mCURE at a PUI

953 Table 1C: Lesson Plan Timeline – MDH Bioinformatics and Hypothesis for mCURE at a 954 Community College

955 Table 1D: Lesson Plan Timeline – MDH Bioinformatics and Hypothesis for mCURE at an R1 956 University

957

958 FIGURES 959 Figure 1. Mindmap of the organization and philosophy of the module.

960

961 SUPPORTING MATERIALS 962 1. Learning Goal Rubrics, LG1 Rubric, LG2 Rubric, LG3 Rubric 963 2. Hypothesis Development and Proposal Module: Parts 1, 2 & 3(for Faculty) and PreClass 964 reading assignments (for Students) 965 3. Powerpoints used in the lesson in a Biochemistry Capstone Course s listed in the 966 timeline table 1A 967 4. An Introduction to Enzymes: Science Behind the Lesson 968 5. Mini-Quizzes used in Lesson 1A 969 6. Papers used for Literature Discussion Components of the lesson 970 7. Grading Rubric for the Draft of the Introduction and Literature Background Component 971 8. Grading Rubric for the first Presentation 972 9. Rubrics for the Peer Review Session 973 10. Powerpoints and Worksheets used in the lesson in an mCURE at a PUI 974 11. Powerpoints and Worksheetsused in the lesson in an mCURE at a Community College 975 12. Powerpoints and Worksheets used in the lesson in an mCURE at an R1 University

976

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977

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