Article Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX
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Unnatural Chemical Biology: Research-Based Laboratory Course Utilizing Genetic Code Expansion Kelsey M. Kean, Kari van Zee, and Ryan A. Mehl* Department of Biochemistry and Biophysics, Oregon State University, 2011 Agriculture and Life Sciences Building, Corvallis, Oregon 97331, United States
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S Supporting Information *
ABSTRACT: The content and design scheme for a readily adaptable, research-based laboratory course in chemical biology are presented. In this course, students interrogate protein structure and function using the site-specific incorporation of noncanonical amino acids by genetic code expansion. The relatively new field of genetic code expansion enables protein engineering with a diverse array of chemical functional groups. In this quarter or semester research-based undergraduate laboratory experience, student teams design, synthesize, and evaluate the structure−function relationships of proteins containing noncanonical amino acids on the basis of a self-selected hypothesis and then communicate their results in a formal manuscript and research presentation. The flexibility and versatility of genetic code expansion and this course structure empower students to engineer novel biomolecules and highlight the use of organic-chemistry principles for interrogating proteins. Generating noncanonical protein variants that have never been produced previously exposes students to novel research and trains them in essential basic biochemistry skills. This researchbased undergraduate laboratory course can easily be adapted, scaled, and implemented on the basis of the interests, demographics, and resources of a given institution. KEYWORDS: Upper-Division Undergraduate, Laboratory Instruction, Inquiry-Based/Discovery Learning, Hands-on Learning/Manipulatives, Proteins/Peptides
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The flexibility and versatility of GCE affords it a unique advantage for a biochemistry or chemical biology course platform. Because GCE exploits the use of a single amber stop codon, TAG, for the incorporation of ncAAs, once the gene for a model protein has been engineered to contain one or more TAG codons, diverse ncAA functionalities can easily be incorporated at this location (Scheme 1). With this approach, a research-based laboratory course was developed focusing on a few key pedagogical concepts: (1) designing and generating new biomolecules stimulates curiosity, fosters personal investment, and obligates controls with rigorous comparative analysis; (2) designing and troubleshooting complex problems in a team increases conceptual understanding; and (3) the planning associated with repeating and improving laboratory experiments is an essential component to solving problems with unknown answers. Herein, a laboratory course is described in which students utilize GCE to incorporate ncAAs into a protein, enabling them to design unique studies, and probe the structures and functions of proteins as part of a genuine research experience. By incorporating GCE into a laboratory course, students are introduced to traditional biochemistry techniques as well as
INTRODUCTION Site-directed mutagenesis is a standard tool for probing the functions of proteins and specific amino acids, but it is traditionally limited to the scope of the 20 natural amino acids. Using genetic code expansion (GCE) for the site-specific incorporation of noncanonical amino acids (ncAAs) greatly expands the biochemistry tool set. Because GCE provides virtually unlimited chemical functionality to probe protein function and structure, it is a powerful chemical biology teaching tool. The growing GCE field also provides the ability to engineer new protein functions, structures, and interactions with other biomolecules or materials.1,2 Despite the increasing applicability and significant research advances being made with chemical biology tools to manipulate biomolecules, little time is spent in traditional lectures and laboratory courses on the ability to robustly engineer proteins with new technologies. Other cutting-edge chemical biology techniques have been successfully integrated into course-based undergraduate research experiences, including synthesizing and characterizing novel small-molecule fluorogenic probes3−5 or naturalproduct-like compounds.6 However, the ability to manipulate biomolecules, especially proteins, in a controlled manner using chemical biology tools such as GCE is far more limited. One example of the successful incorporation of GCE into an undergraduate teaching laboratory course was a 3 week module based on manipulating the spectral properties of green fluorescent protein (GFP) with ncAAs.7 © XXXX American Chemical Society and Division of Chemical Education, Inc.
Received: January 4, 2018 Revised: August 31, 2018
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DOI: 10.1021/acs.jchemed.8b00011 J. Chem. Educ. XXXX, XXX, XXX−XXX
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Scheme 1. Overview of GCE Componentsa
a Each engineered GCE RS−tRNA pair can site-specifically incorporate an ncAA into a protein in response to a TAG codon. When the ncAA is withheld, a protein truncated at the TAG site results. Elongation factor Tu (EF-Tu) is endogenous to the host, E. coli.
Next, orthogonal pairs are evolved to efficiently incorporate an ncAA of interest but not incorporate natural amino acids, and they are evaluated for their efficiency in producing ncAAmutant proteins and fidelity in not incorporating natural amino acids.8 Once established, a standard GCE system is composed of four key components (Scheme 1): (1) the ncAA to sitespecifically incorporate into a protein, (2) the orthogonal aminoacyl-tRNA synthetase (RS), (3) the orthogonal tRNA, and (4) the gene of interest with a TAG codon at the site for ncAA incorporation. GCE currently also relies on using ribosomes and elongation factors (including elongation factor EF-Tu) endogenous to E. coli. The design of the optimized system allows for versatility in generating new ncAA-mutant proteins. The genes for an RS−tRNA pair are combined onto a single GCE plasmid, which can easily be paired up with any protein-expression plasmid containing a platform protein gene with a TAG codon site. Although many biochemistry courses explore the effects of natural amino acid mutations on protein function, GCE affords a few practical and pedagogical advantages. One advantage for students when designing their studies is the ability to exponentially increase the number of ncAA-mutant protein options as each TAG-interrupted gene and RS−tRNA pair is added to the tool set. For example, in this course, six different TAG codons were selected and incorporated into each of the platform protein genes (CA and HPII). Each of these different TAG-interrupted genes were combined in a cell with one of three different GCE plasmids allowing for 18 unique protein options for each platform protein. Additionally, an evolved orthogonal RS−tRNA pair can often incorporate multiple different but related ncAAs, such as para-substituted phenylalanines. This characteristic (named the permissivity) of an RS−tRNA pair expands the versatility of this method even more because a single GCE plasmid system can incorporate a large variety of ncAAs depending on which ncAA is provided in the culture.8−12 Taking permissivity into account, seven different amino acids could be incorporated (while requiring only three RS−tRNA pairs), allowing for 42 different options for each platform protein. With these combinatorial effects, a small set of cell stocks easily provides many options to students with diverse ways to probe proteins with minimal additional setup work for instructors. The most valuable educational advantages of GCE come from the use of these translational tools and the availability of
trained in next-generation chemical biology technology. This combination provides undergraduates with an understanding that they are capable of manipulating biological systems as they move forward into their careers. For this course, the instructor selects the protein and prepares a set of mutants enabling ncAA incorporation at a variety of locations in that platform protein. Each team’s project is guided by a hypothesis it develops and experiments it selects and designs on its own. This course emphasizes execution and understanding of basic practical skills typical of a biochemistry laboratory course, such as recombinant protein expression, purification, characterization by SDS-PAGE, and kinetic analysis. It also promotes hypothesis and experiment development, understanding of the central dogma, the ability to probe and assess structure− function relationships, critical thinking, the use and assessment of primary literature, and the development of scientific writing and communication skills. An intellectual overview of the curriculum is presented, and sample student results and suggestions for implementation and adaptation at other institutions are provided. The version of the research-based laboratory course presented herein currently uses GCE to serve over 90 upper-level students from diverse academic backgrounds, such as biochemistry, bioengineering, biology, and chemistry. Key aspects of pedagogy and strategies for tuning the project focus to suit the research interests of the program (for example, mechanistic enzymology, surface immobilization, drug development, structural probes, or protein interactions) are highlighted. Although any protein of interest to the instructor can be used, two proteins, human carbonic anhydrase II (CA) and Escherichia coli (E. coli) catalase HPII (HPII), are described in detail as platform proteins. The full laboratory manual, instructor guide, supporting course materials, and all genetic constructs and plasmids are available to interested instructors (Supporting Information).
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EDUCATIONAL ADVANTAGES OF TEACHING WITH GENETIC CODE EXPANSION Central to GCE technology is an engineered aminoacyl-tRNA synthetase−tRNA pair (RS−tRNA) that encodes an ncAA in response to an amber stop codon, TAG, in a gene of interest (Scheme 1). Initially, the RS−tRNA pairs are evolved to be orthogonal to the translational components of the cell to ensure no cross reactivity with canonical RS−tRNA pairs. B
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COURSE EXPERIMENTAL OVERVIEW AND ADAPTABILITY Traditional biochemistry protein techniques and skills are at the foundation of this course, making it relatively easy to implement. In addition, this course design emphasizes research hypothesis development, the generation of new biomolecules, and solving scientific problems as a team. Students described their scientific findings in the context of the literature and presented them both as a group oral presentation and individually as a scientific manuscript (Box 1). Comprehensively, this project ownership stimulated curiosity and fostered personal investment in their projects.
new amino acid chemistry. The use of altered translational components provides firsthand understanding that scientists are not limited to nature’s fundamental building blocks. This synthetic biology component opens students’ minds to the many ways biomolecules can be engineered and requires students to deeply understand the chemical steps of translation. In undergraduate training, students rarely have an opportunity to study biomolecules that no one has yet explored. While the amino acid sites on these platform proteins may have been studied in the literature using natural amino acids, students are the first to probe these sites using ncAAs, so the outcomes are unknown. This component energizes the course with scientific curiosity and makes it absolutely clear how important control experiments are for the results to have meaning. The manipulation of a natural amino acid to an ncAA forces students to dig back into organic chemistry knowledge regarding pKa, hydrogen bonding, organic functional group size, electrostatic interactions, and reactivity in order to understand the properties of each new ncAA (Figure 1). Students also are able to design
Box 1. Intended Learning Outcomes • Become literate in specialized language and fundamental concepts of chemical biology and biochemistry. • Collect, accurately record, analyze, and interpret experimental data. • Develop an awareness of the ability to generate and manipulate biomolecules using nonstandard building blocks. • Be able to design, plan, implement, and manage interdependent experiments. • Navigate, utilize, and critically assess scientific literature and place the research in the context of existing literature. • Clearly and effectively communicate science to a scientific audience. The course design is focused on a platform protein, referred to as the wild type, which the students modified with different ncAAs. The course has been vetted with as few as two and as many as six different platform proteins per term that meet specific characteristic criteria (Figure 2). Other proteins that fit these criteria could readily be implemented. As example model platform proteins, both CA and HPII (Figure 2) are well studied and well characterized with many
Figure 1. Noncanonical amino acids that impart unique functions with specific applications: example ncAAs used in this course.
studies that probe interactions not possible with natural amino acids. For example, tyrosine amino acids are common key ligands for cofactors and are often found in active sites and at interfaces. The ncAA set focuses on tyrosine mimics and the ability to alter the para-hydroxyl functionality without altering the aromatic scaffold. This allows students to design studies not possible with natural amino acids, for example, altering the axial tyrosine heme ligand or the tyrosine tunnel gate in catalases.13 In addition, new ncAA functionalities can be exploited as spectroscopic probes or for attaching functionality with bioorthogonal ligations, allowing for new methods to probe the proteins or alter their structure and function. In particular, this course is well-suited for studying structure− function relationships, protein evolution, and protein engineering. Additional amino acid sites for incorporation, different ncAAs, and platform proteins can be readily implemented in this course according to the demographics and interests of the students and instructors and the desired outcomes of the class.
Figure 2. Characteristics of model proteins. Genetic information for model proteins CA and HPII are provided in the Supporting Information and the Laboratory Manual.35 C
DOI: 10.1021/acs.jchemed.8b00011 J. Chem. Educ. XXXX, XXX, XXX−XXX
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Figure 3. Experimental overview and timeline integrated with the key pedagogical components and experimental details.
typically taught by one instructor with the assistance of one graduate or undergraduate teaching assistant. The laboratory course also had one 50 min discussion-based class session per week. This period was used to discuss theory for key steps, tips for success, hypothesis development, literature findings, and methods for analysis of experimental data. An overview of this research-based laboratory course integrated with the key pedagogical components and timeline is shown in Figure 3. A detailed weekly overview is presented in the Supporting Information (Table S2, p S7). The course was pitched to the undergraduate teams as a research project that was handled much like a graduate student rotation. By this point in their undergraduate training, the students had experienced many modular laboratory courses focused on learning laboratory techniques. They embraced the relatively new idea of not following a list of instructions that arrived at a preplanned result. The students were informed they would be totally independent and the success of the project relied only on their team’s organization, preparation, and problem-solving skills. In reality, the course was structured with clear tasks, goals, and deliverables every week to keep the teams on target. In our experience, it was key to provide sufficient room for failure in each of the experimental steps as this was essential to learning how to perform independent science. There were ample opportunities for teams to discuss and implement more effective experimental designs in subsequent weeks, removing the burden from instructors that every experiment must work (highlighted in Figure 3).
structures available in the Protein Data Bank (PDB), providing students with significant background information upon which to build their studies. Both proteins have industrial applications (e.g., CA in carbon sequestration and HPII in fabric production) and biomedical applications (e.g., CA in artificial lungs and HPII in aging), making them interesting to students with diverse career and academic interests. Both proteins have high expression levels; are soluble and stable; and have relatively simple, inexpensive activity assays requiring basic equipment, making them amenable to a larger volume teaching laboratory environment. The activity of CA was measured using a para-nitrophenyl acetate hydrolysis assay requiring only a spectrophotometer. The activity of HPII was measured by monitoring oxygen production and required a gas-pressure or oxygen sensor. Although the focus is on these model proteins, this course has been successfully carried out using T4 lysozyme, nitroreductase, malate dehydrogenase, triosphosphate isomerase, aspartate amino transferase, and histidinol dehydrogenase (Supporting Information, Table S6, pp S46−S47). This course was implemented for 14 years on a small scale (20 students per offering) at a primarily undergraduate institution (8 years) and on a large scale (90 students per offering) at a large, public research university (6 years) for a total of 700 students. The independent research nature of this course lent itself well to upper-division undergraduates students who were concurrently taking the biochemistry lecture or had already completed this series. The course is highly adaptable, with successful implementation in 15 week semester format and 10 week quarter format courses. Herein, the course is presented in a 10 week format delivered at a large, public research university with four sections of 24 students per section. Within a single section, students were divided into teams of three to four by the instructor. Each section met for 3 h twice a week and was
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HAZARDS
Students, instructors, and teaching assistants were required to complete university safety training before entering the laboratory. Safety googles, lab coats, and gloves were to be worn at all times in the laboratory. E. coli is a BSL-1 organism, and the appropriate precautions should be taken. The main D
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Figure 4. Sample results from student manuscripts and time frame of experiments. (A) Sample figure generated using Pymol14 detailing a hypothesis probing Tyr415, which forms covalent bonds with heme, and His392, which is essential for folding, in HPII. (B) Sample SDS-PAGE gel of purified HPII (∼84 kDa molecular weight) showing the wild-type protein (WT), HPII with 3-nitrotyrosine incorporated at site 415 (+3-NY), HPII with para-aminophenylalanine incorporated at site 415 (+pAF), and the corresponding negative controls of the HPII mutants expressed in the absence of ncAAs (−3-NY and −pAF). The molecular weights of the ladder are labeled (kDa). As can be seen by comparing +3-NY and +pAF, not all TAG site−ncAA combinations express equally well, because some sites are critical for protein folding and function. (C) Sample kinetic characterization of wild-type CA, CA with para-bromophenylalanine incorporated at site 20 (pBrF), and CA with O-methyltyrosine incorporated at site 20 (O-CH3Y) and compared with the wild-type literature values where available. For the Lineweaver−Burk plot in this sample, error bars were less than 10% in all cases and are not shown. (D) Sample results of a unique assay probing the activity at increased temperature of wild-type CA, CA with 4-BrF incorporated at site 20 (4-BrF), and CA with O-CH3Y incorporated at site 20 (O-CH3Y).
and implemented the key study the team designed on the basis of its hypothesis (Figure 4D). The term culminated in each team giving a presentation of its research and each individual submitting a final scientific manuscript. Over the 10 week term, students were guided to convert concepts and data into a Biochemistry-style manuscript and research presentation containing quality figures. To facilitate this process, students completed a series of structured laboratory deliverables and two individual manuscript drafts (see Supporting Information pp S22−S43 for the laboratory deliverables, and pp S48−S49 for sample student manuscripts). This cycle of having the students generate components of the final products and obtaining timely, constructive feedback helped the students use the literature as a model for their work and refine their experimental designs and data collection. Initial student results focused on an introduction describing the key chemical features the teams explored with their hypotheses. Molecular detail was required to highlight the molecular interactions under investigation, allowing students to develop ChemDraw and Pymol14 skills (Figure 4A). During class, student teams debated the quality, clarity, and testability of their hypotheses. A second phase focused on preparing essential protein expression and purification information that enabled others to replicate their experiments. Clear depictions
chemical hazards are acrylamide (used for SDS-PAGE), which is a potential neurotoxin; acetonitrile (used for the PNPA assay), which is an irritant and highly flammable; and 4nitrophenol (a product of the PNPA assay), which is an acute toxin and should be handled with care. Complete lists of all chemicals used in each laboratory activity with CAS numbers and associated hazard risks are provided in Supporting Information (Instructor Guide, Table S5, p S17).
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COURSE OUTLINE AND EXAMPLE OF STUDENT RESULTS Week 1 focused on learning about the assigned platform protein and developing a hypothesis (Figure 4A). Week 2 focused on finishing preliminary hypothesis development and corresponding experiments, expressing wild-type protein, and selecting ncAAs and sites of incorporation. Week 3 involved purifying the wild-type protein and expressing ncAA-mutant proteins with appropriate controls (Figure 4B). Week 4 entailed purifying ncAA-mutant proteins and crudely testing the activity of the wild-type protein. During week 5, the teams designed kinetic assays on the basis of the literature and began characterizing the function of the wild-type protein (Figure 4C). During weeks 6−9, each team continued to determine kinetic constants of the wild-type and ncAA-mutant proteins E
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Table 1. Comparison of Student Responses to Assessment of Course Learning Outcomes Statements for Response
Student Responses by Category, % (N = 89) Strongly Disagree
Disagree
Neither Agree nor Disagree
Agree
Strongly Agree
This course has improved my understanding of specialized language and fundamental concepts 0 1 7 49 43 of chemical biology and biochemistry. This course has improved my ability to design, generate, and characterize new proteins. 0 2 7 44 47 This course has improved my ability to collect, accurately record, analyze, and interpret 0 2 18 51 29 experimental data. This course has increased my awareness of how to manipulate biomolecules using nonstandard 0 2 11 33 54 building blocks. This course has improved my ability to design, plan, and manage interdependent experiments. 0 3 11 51 34 This course has improved my ability to navigate, utilize, and critically assess scientific literature. 0 7 17 42 34 This course has improved my ability to communicate clearly and effectively science to a 0 2 17 51 29 scientific audience. By modifying proteins with noncanonical amino acids instead of natural amino acids, this course required me to develop a deeper understanding of fundamental components from my past course work in: Translation 0 3 29 49 18 Protein structure 0 3 15 39 43 Enzyme kinetics 0 0 14 30 56 Organic chemistry 2 9 31 46 11
skills that will serve them along any path, especially in science. The students also knew that when they interview for jobs, graduate schools, or professional schools, they may need to describe a meaningful research experience, how they solved unexpected problems, and how they managed working in a team. The intended learning outcomes (Box 1) were assessed with anonymous student surveys at the end of the 10 week course (Table 1). The vast majority of the students agreed or strongly agreed that the course met each intended learning outcome.
of protein purification and analysis in labeled SDS-PAGE images (Figure 4B) were emphasized in laboratory deliverables and manuscript drafts. The third phase was a nontrivial process involving the processing of raw enzyme kinetic data into representative figures and clear data tables with error bars (Figure 4C). One lecture was dedicated to how a kinetic model might not match experimental data because of the experimental assumptions inherent to that model. Again, the literature on the platform proteins was used for comparison of kinetic results and clear presentation and interpretation of data. Obtaining results that focused on the team’s hypothesis (Figure 4D) was the final phase. The teams presented their entire research projects to the class, which provided a valuable peer-review quality control step. Presentations were scheduled before the due date of the final manuscript, allowing students to address constructive criticism on how to present these data to focus readers’ attention on key differences and findings.
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Faculty-Centered Outcomes and Benefits
Although often overlooked, incorporating a research experience, especially one based around the current research interests of a faculty member, into a credit-bearing course is also beneficial to the faculty member. In agreement with our own experience, faculty members who develop and teach their own course-based undergraduate research experiences report many benefits, including being able to connect their teaching and research.24 Additionally, incorporating the research interest of a faculty member into a course can provide the more tangible benefit of directly contributing to their research program and can serve as a recruitment tool for finding capable and motivated undergraduate students.24 After completing the course, successful students can utilize more expensive ncAAs and add and screen additional amino acid sites. In some cases, students from this course continued their projects, which resulted in publication.25 Although a preformed course design focused on GCE of the proteins CA and HPII was presented, this course can easily be modified by involved faculty members to accommodate their research proteins and interests in, for example, structure−function relationships, material science, or protein evolution. This course provides the positive benefits that faculty members find from integrating their roles as educators and researchers without a high energy barrier in terms of design and setup.
OUTCOMES AND BENEFITS
Student-Centered Outcomes and Benefits
Incorporating an undergraduate research experience into a course has been extensively shown to positively benefit students in a number of ways, including by improving conceptual understanding, 15−17 increasing self-confidence,15,17−20 improving critical-thinking skills,19,21,22 improving scientific communication,15−21 providing a deeper understanding of the nature of scientific research,17,19,22,23 and increasing students’ ability to handle and overcome setbacks and uncertainty.17,18,20 On the basis of observations by the instructor team and student evaluations, students have indeed reaped these benefits in this research-based laboratory course. Although some faculty have found that such courses can be met with student resistance because of the amount of experimental uncertainty,24 in our experience, students have risen to the occasion and, in many cases, have provided final manuscripts of publication quality and with truly novel and interesting results. In its current form, this course served as a capstone course for upper-division undergraduates and required students to remember and utilize the biology, chemistry, biochemistry, and engineering they learned over their educational careers while providing them with valuable
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PRACTICAL SUGGESTIONS FOR INSTRUCTORS This course has four notable challenges that should be considered before implementation: (1) the logistics of expressing many cultures and purifying many proteins simultaneously; (2) the instruments and supplies needed to F
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induce gene expression. The gene of interest should be encoded on a pET or pBAD plasmid and paired with a pDule-1 or pDule-2 plasmid containing the GCE machinery. This plasmid combination in BL21-AI or DH10B cells results in a reliable ratio of tRNA, RS, and mRNA transcript for efficient ncAA-mutant protein production with high fidelity. Fortunately, some of the best functioning GCE machinery also works with inexpensive ncAAs. It is recommended that proteins are engineered with Cterminal His-tags, so teams only purify full-length protein. The location of the TAG site in a gene will produce a ratio of fulllength ncAA-mutant protein to protein truncated at the TAG site because cellular release factor 1 competes with the orthogonal charged tRNA. Although truncated protein is often detectable in SDS-PAGE analysis of crude cell expressions, a C-terminal His-tag ensures only full length ncAA-mutant protein is purified. An additional consideration to increase ncAA-mutant protein yield is the genetic origin. Thus far, every protein that has been cloned directly from the E. coli genome (without codon optimization) has expressed robustly. Alternatively, the genetic construct can be codon optimized for expression in E. coli or cloned from a prokaryotic source. When following these guidelines for selecting genes and expression format, the major issue with identifying proteins for the course has not been ncAA-mutant protein yield but the availability of a cost-effective, robust kinetic assay for characterizing protein function (Table S6, pp S46−S47).
run many kinetic assays simultaneously; (3) the selection of proteins, GCE components, ncAA sites for incorporation, and (4) the cost of the ncAAs. An Instructor Guide is available in the Supporting Information containing detailed sections on logistical challenges for the scale of the course offering and time management. Scaling Considerations for Implementation
For the first 9 years, this course was taught at Franklin and Marshall College (F&M) to a class of ∼20 undergraduates in a single, 4 h laboratory per week. This format allowed each group of four students to express five 50 mL cultures in one large shaker and lyse cells with sonication without logistical problems. The small class size at F&M and the availability of a UV−vis instrument for each group allowed each group to study a different protein. Although this required an upfront cost to clone six genes and generate TAG sites in each, the cost of reagents for the six enzyme assays was reasonable, and the logistics of organizing and troubleshooting with six groups was manageable (Table S6, pp S46−S47). In transitioning this laboratory to its current form at Oregon State University, in which students attend two 3 h laboratory sessions and one 1 h lecture session per week, logistical challenges arose, particularly as the course expanded to include up to 96 students and four different instructors over the last 5 years. The largest constraint was access to instrumentation and reagents for enzyme assays, so the course was minimized to include only the two enzymes with the cheapest and most versatile assays that did not require the same instrumentation: CA and HPII. Expressing 75−100 different cultures in a single week required adding shaker space, and lysing many cell types was expedited by the acquisition of a microfluidizer.
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CONCLUSION
The design of an entire chemical biology laboratory course was presented, utilizing GCE in order to provide students with the opportunity to conduct novel research within the constraints of a 10 week term. Course-based undergraduate research is quickly becoming an essential part of undergraduate education as it overcomes many of barriers preventing students from doing research and allows a greater number of students to carry out scientific research within a classroom setting. However, designing a new course, especially a research-based laboratory course, can be resource intensive to develop, both in terms of instructor time and cost. Although course-based undergraduate research experiences in chemical biology are growing,28,29 these courses tend to focus around the synthesis, manipulation, and characterization of small molecules and their interactions and functions.3−5,30−32 Courses in which biomolecules, in particular proteins, are manipulated using chemical biology tools are far more limited,33,34 and very few utilize technologies such as GCE, which allow for controlled and directed manipulation and synthesis of novel proteins.7 In this course, students generated and evaluated new biomolecules and, in the process, deepened their understanding of biochemistry and chemical biology and benefited beyond the laboratory classroom in measurable ways. This course can be readily adapted to meet the needs of different instructors, student populations, interests, skill levels, and resources. We are providing this chemical biology course, laboratory manual,35 instructor guide, and genetic components with all of our resources and course materials for use at other institutions to expand the exposure of undergraduates to chemical biology and research-based learning.
Genetic Code Expansion Steps to Success
The exciting science for the student teams comes from characterizing and comparing the abilities of ncAA-mutant and wild-type proteins, so the steps for generating pure ncAAmutant proteins must be robust. To enable success, the platform proteins and GCE components were selected for low technical challenges and high expression yields. On the basis of the ease of use and versatility, some GCE tools are better suited for use in an undergraduate laboratory setting than others. However, the GCE field is growing exponentially with regular reports of new and improved tools.2,26 Because it is never obvious which genetic constructs will enable high ncAA-mutant protein production in E. coli when paired with GCE machinery, some key steps to success are provided. The most important factor is how well the wild-type protein expresses in E. coli; every platform protein used in this course produced over 100 mg of wild-type protein per liter of media. When these expression constructs were paired with the GCE systems described here, they produced over 50 mg of ncAA-mutant protein per liter of media. The components to consider in order to reach these levels are (1) plasmid type, (2) cell type, (3) media type, and (4) ncAA selection. Although many different GCE systems have been reported to work in a variety of conditions,2,26 robust ncAA-mutant protein expression results from a balanced expression rate that is maximized with specific pairings. The autoinduction media described here27 should be used as it provides a slow rate and a long expression time for ncAA-mutant protein production, which is forgiving to GCE machinery that cannot keep up with fast expression conditions. It also eliminates the need for students to come into lab between scheduled lab periods to G
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(9) Young, D. D.; Young, T. S.; Jahnz, M.; Ahmad, I.; Spraggon, G.; Schultz, P. G. An Evolved Aminoacyl-tRNA Synthetase with Atypical Polysubstrate Specificity. Biochemistry 2011, 50 (11), 1894−1900. (10) Young, D. D.; Jockush, S.; Turro, N. J.; Schultz, P. G. Synthetase polyspecificity as a tool to modulate protein function. Bioorg. Med. Chem. Lett. 2011, 21 (24), 7502−7504. (11) Stokes, A. L.; Miyake-Stoner, S. J.; Peeler, J. C.; Nguyen, D. P.; Hammer, R. P.; Mehl, R. A. Enhancing the utility of unnatural amino acid synthetases by manipulating broad substrate specificity. Mol. BioSyst. 2009, 5 (9), 1032−1038. (12) Miyake-Stoner, S. J.; Refakis, C. A.; Hammill, J. T.; Lusic, H.; Hazen, J. L.; Deiters, A.; Mehl, R. A. Generating Permissive SiteSpecific Unnatural Aminoacyl-tRNA Synthetases. Biochemistry 2010, 49 (8), 1667−1677. (13) Diaz, A.; Loewen, P. C.; Fita, I.; Carpena, X. Thirty years of heme catalases structural biology. Arch. Biochem. Biophys. 2012, 525 (2), 102−110. (14) PyMOL Molecular Graphics System, Version 1.3; Schrödinger, LLC: San Carlos, CA, 2010. (15) Kardash, C. M. Evaluation of an undergraduate research experience: Perceptions of undergraduate interns and their faculty mentors. J. Educ. Psychol. 2000, 92 (1), 191−201. (16) Seymour, E.; Hunter, A.-B.; Laursen, S. L.; DeAntoni, T. Establishing the benefits of research experiences for undergraduates in the sciences: First findings from a three-year study. Sci. Educ. 2004, 88 (4), 493−534. (17) Hunter, A. B.; Laursen, S. L.; Seymour, E. Becoming a scientist: The role of undergraduate research in students’ cognitive, personal, and professional development. Sci. Educ. 2007, 91 (1), 36−74. (18) Lopatto, D. Survey of Undergraduate Research Experiences (SURE): First Findings. Cell Biol. Educ. 2004, 3 (4), 270−277. (19) Thiry, H.; Laursen, S. L.; Hunter, A. B. What Experiences Help Students Become Scientists? A Comparative Study of Research and Other Sources of Personal and Professional Gains for STEM Undergraduates. J. Higher Educ. 2011, 82 (4), 357−388. (20) Ward, C.; Bennett, J. S.; Bauer, K. W. Content analysis of undergraduate research student evaluations, 2003. University of Delaware. www1.udel.edu/RAIRE/Content.pdf (accessed July 25, 2018). (21) Bauer, K. W.; Bennett, J. S. Alumni perceptions used to assess undergraduate research experience. J. High. Educ. 2003, 74 (2), 210− 230. (22) Gasper, B. J.; Gardner, S. M. Engaging Students in Authentic Microbiology Research in an Introductory Biology Laboratory Course is Correlated with Gains in Student Understanding of the Nature of Authentic Research and Critical Thinking. J. Microbiol. Biol. Educ. 2013, 14 (1), 25−34. (23) Ryder, J.; Leach, J. University science students’ experiences of investigative project work and their images of science. Int. J. Sci. Educ. 1999, 21 (9), 945−956. (24) Shortlidge, E. E.; Bangera, G.; Brownell, S. E. Faculty Perspectives on Developing and Teaching Course-Based Undergraduate Research Experiences. BioScience 2016, 66 (1), 54−62. (25) Jackson, J. C.; Duffy, S. P.; Hess, K. R.; Mehl, R. A. Improving nature’s enzyme active site with genetically encoded unnatural amino acids. J. Am. Chem. Soc. 2006, 128 (34), 11124−11127. (26) Xiao, H.; Schultz, P. G. At the Interface of Chemical and Biological Synthesis: An Expanded Genetic Code. Cold Spring Harbor Perspect. Biol. 2016, 8 (9), No. a023945. (27) Hammill, J. T.; Miyake-Stoner, S.; Hazen, J. L.; Jackson, J. C.; Mehl, R. A. Preparation of site-specifically labeled fluorinated proteins for 19F-NMR structural characterization. Nat. Protoc. 2007, 2 (10), 2601−2607. (28) Heemstra, J. M.; Waterman, R.; Antos, J. M.; Beuning, P. J.; Bur, S. K.; Columbus, L.; Feig, A. L.; Fuller, A. A.; Gillmore, J. G.; Leconte, A. M.; Londergan, C. H.; Pomerantz, W. C. K.; Prescher, J. A.; Stanley, L. M. Throwing Away the Cookbook: Implementing Course-Based Undergraduate Research Experiences (CUREs) in Chemistry. In Educational and Outreach Projects from the Cottrell
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.8b00011.
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Instructor guide containing information for instructors and teaching assistants, information on all plasmids, complete list of chemical hazards, minimum equipment needs, lecture and laboratory deliverables, and sample student manuscripts (PDF, DOCX)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Ryan A. Mehl: 0000-0003-2932-4941 Notes
The authors declare no competing financial interest. The laboratory manual is provided as an open-source textbook through Oregon State University (http://library.open. oregonstate.edu/chembiolab/). Plasmids are available in the Addgene plasmid repository (https://www.addgene.org/).
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ACKNOWLEDGMENTS The authors would like to thank Rachel Henson and Ramya Raman for thoughtful conversations and assistance with revising the laboratory manual and Joseph Porter, Sarah Clark, and Ian Abbene for assistance with figure design. This work was supported in part by a grant from the National Science Foundation (MCB-1518265) to R.A.M.
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REFERENCES
(1) Young, D. D.; Schultz, P. G. Playing with the Molecules of Life. ACS Chem. Biol. 2018, 13 (4), 854−870. (2) Dumas, A.; Lercher, L.; Spicer, C. D.; Davis, B. G. Designing logical codon reassignment - Expanding the chemistry in biology. Chem. Sci. 2015, 6 (1), 50−69. (3) Johnson, R. J.; Savas, C. J.; Kartje, Z.; Hoops, G. C. Rapid and Adaptable Measurement of Protein Thermal Stability by Differential Scanning Fluorimetry: Updating a Common Biochemical Laboratory Experiment. J. Chem. Educ. 2014, 91 (7), 1077−1080. (4) Kowalski, J. R.; Hoops, G. C.; Johnson, R. J. Implementation of a Collaborative Series of Classroom-Based Undergraduate Research Experiences Spanning Chemical Biology, Biochemistry, and Neurobiology. CBE Life Sci. Educ. 2016, 15 (4), No. ar55. (5) Johnson, R. J.; Hoops, G. C.; Savas, C. J.; Kartje, Z.; Lavis, L. D. A Sensitive and Robust Enzyme Kinetic Experiment Using Microplates and Fluorogenic Ester Substrates. J. Chem. Educ. 2015, 92 (2), 385−388. (6) McKenzie, N.; McNulty, J.; McLeod, D.; McFadden, M.; Balachandran, N. Synthesizing Novel Anthraquinone Natural Product-like Compounds To Investigate Protein−Ligand Interactions in Both an in Vitro and in Vivo Assay: An Integrated Research-Based Third-Year Chemical Biology Laboratory Course. J. Chem. Educ. 2012, 89 (6), 743−749. (7) Maza, J. C.; Villa, J. K.; Landino, L. M.; Young, D. D. Utilizing Unnatural Amino Acids To Illustrate Protein Structure-Function Relationships: An Experiment Designed for an Undergraduate Biochemistry Laboratory. J. Chem. Educ. 2016, 93 (4), 767−771. (8) Cooley, R. B.; Karplus, P. A.; Mehl, R. A. Gleaning unexpected fruits from hard-won synthetases: probing principles of permissivity in non-canonical amino acid-tRNA synthetases. ChemBioChem 2014, 15 (12), 1810−1819. H
DOI: 10.1021/acs.jchemed.8b00011 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
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Scholars Collaborative Undergraduate and Graduate Education Vol. 1; Waterman, R.; Feig, A., Eds.; ACS Symposium Series 1248; American Chemical Society: Washington, DC, 2017; pp 33−63. (29) Van Dyke, A. R.; Gatazka, D. H.; Hanania, M. M. Innovations in Undergraduate Chemical Biology Education. ACS Chem. Biol. 2018, 13 (1), 26−35. (30) Pontrello, J. K. Metalloprotease Peptide Inhibitors: A SemesterLong Organic Synthetic Research Project for the Introductory Laboratory Course. J. Chem. Educ. 2015, 92 (5), 811−818. (31) Bascom-Slack, C. A.; Arnold, A. E.; Strobel, S. A. StudentDirected Discovery of the Plant Microbiome and Its Products. Science 2012, 338 (6106), 485−486. (32) Li, Y. F.; Tsai, K. J. S.; Harvey, C. J. B.; Li, J. J.; Ary, B. E.; Berlew, E. E.; Boehman, B. L.; Findley, D. M.; Friant, A. G.; Gardner, C. A.; Gould, M. P.; Ha, J. H.; Lilley, B. K.; McKinstry, E. L.; Nawal, S.; Parry, R. C.; Rothchild, K. W.; Silbert, S. D.; Tentilucci, M. D.; Thurston, A. M.; Wai, R. B.; Yoon, Y. J.; Aiyar, R. S.; Medema, M. H.; Hillenmeyer, M. E.; Charkoudian, L. K. Comprehensive curation and analysis of fungal biosynthetic gene clusters of published natural products. Fungal Genet. Biol. 2016, 89, 18−28. (33) Van Dyke, A. R.; Etemad, L. S.; Vessicchio, M. J.; Naclerio, G. A.; Jedson, V. Capture-Tag-Release: A Strategy for Small Molecule Labeling of Native Enzymes. ChemBioChem 2016, 17 (17), 1602− 1605. (34) Champasa, K.; Longwell, S. A.; Eldridge, A. M.; Stemmler, E. A.; Dube, D. H. Targeted identification of glycosylated proteins in the gastric pathogen Helicobacter pylori (Hp). Mol. Cell. Proteomics 2013, 12 (9), 2568−2586. (35) Mehl, R.; van Zee, K.; Kean, K. Chemical Biology & Biochemistry Laboratory Using Genetic Code Expansion Manual; Pressbooks, 2018. Available at http://library.open.oregonstate.edu/chembiolab/.
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DOI: 10.1021/acs.jchemed.8b00011 J. Chem. Educ. XXXX, XXX, XXX−XXX
