Site-Directed Mutagenesis Study of an Antibiotic-Sensing Noncoding

May 10, 2017 - Site-Directed Mutagenesis Study of an Antibiotic-Sensing Noncoding RNA Integrated into a One-Semester Project-Based Biochemistry Lab ...
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Site-Directed Mutagenesis Study of an Antibiotic-Sensing Noncoding RNA Integrated into a One-Semester Project-Based Biochemistry Lab Course Timea Gerczei* Department of Chemistry, Ball State University, Muncie, Indiana 47306, United States S Supporting Information *

ABSTRACT: A laboratory sequence is described that is suitable for upper-level biochemistry or molecular biology laboratories that combines project-based and traditional laboratory experiments. In the project-based sequence, the individual laboratory experiments are thematically linked and aim to show how a bacterial antibiotic sensing noncoding RNA (the ykkCD riboswitch) recognizes its target antibiotic, tetracycline. The novelty of the curriculum lies in its extensive coverage of bioinformatics as well as its focus on synthesis, purification, and functional studies of a biologically active RNA. Further advantage of the curriculum is that the model system, the ykkCD riboswitch, regulates expression of a gene that is involved in bacterial antibiotic resistance. Hence, the project makes this important issue part of the undergraduate biochemistry curriculum. The curriculum is available in Open Access format as part of a biochemistry laboratory manual by Gerczei and Pattison. This manuscript summarizes the curriculum and provides suggestions to the instructor regarding its implementation. KEYWORDS: Upper-Division Undergraduate, Biochemistry, Laboratory Instruction, Inquiry-Based/Discovery Learning, Molecular Biology

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biochemistry laboratories;9 and (iii) the curriculum raises awareness of an important issue, bacterial antibiotic resistance, by investigating the specificity of an antibiotic sensor RNA.

espite the documented advantage of inquiry or problembased pedagogies in developing higher-order cognitive skills,1 a survey of institutions offering ACS accredited chemistry degrees revealed that only 8% of these institutions incorporated these nontraditional methods in the general chemistry curriculum.2 SciFinder searches using the terms “inquiry-based” or “problem-based” biochemistry lab found 16 project-based lab sequences. Three of the lab sequences contained a combination of skill-builder (expository) and project-based modules;3 the rest of them were purely projectbased. The majority of these lab sequences investigated a protein (protein isolation, purification, kinetic studies),4 while the others contained a significant molecular biology component (PCR, primer design, restriction digest, etc.).3a,5 Only two lab sequences investigated RNA,6 even though during the past 10 years, the view of RNA as a biologically active molecule drastically changed.7 RNA is transformed from a mere carrier of genetic information to a biologically active molecule that has enzymatic activity and the power to regulate cellular processes.8 The novelty of the work described below is threefold: (i) the curriculum strikes a good balance between traditional and nontraditional teaching methods; (ii) it covers the majority of topics recommended by ACS and ASBMB for introductory © XXXX American Chemical Society and Division of Chemical Education, Inc.

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BACKGROUND AND SIGNIFICANCE OF THE RESEARCH PROJECT USED BY THIS CURRICULUM In the biochemistry lab curriculum presented here, inquirybased learning is achieved by engaging students in a hypothesisdriven, real-life research project: students generate point mutations in an antibiotic-sensing, noncoding RNA (the ykkCD riboswitch10) and evaluate how those mutations affect the ability of the ykkCD riboswitch to recognize the antibiotic tetracycline (Figure 1A).11 Tetracycline binding to the ykkCD riboswitch unfolds a transcription terminator stem and thus enables transcription of the ykkCD efflux pump mRNA (Gerczei et al., unpublished). As a result, the efflux pump is synthesized and able to transport a wide variety of toxic compounds (like tetracycline) out of the cell. Received: July 31, 2016 Revised: April 4, 2017

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Figure 1. YkkCD toxin sensor appears to recognize the antibiotic tetracycline. (A) Schematics of the ON and OFF state of the ykkCD tetracycline sensor riboswitch. Riboswitches are short RNAs that specifically recognize a small molecule and regulate gene expression by allosteric structural change. They are located in the 5′ UTR of the gene they regulate. In the absence of tetracycline, the ykkCD riboswitch folds into a structure that prevents transcription of a multidrug-resistance efflux pump gene (MDR pump). MDR pump ORF stands for MDR pump open reading frame. Once tetracycline is bound to the ykkCD riboswitch, a conformation change takes place that allows for production of the MDR pump. (B) Tetracycline binding to the ykkCD sensor RNA is monitored via quenching of tetracycline fluorescence. The ykkCD riboswitch specifically recognizes the antibiotic tetracycline. Fluorescence quenching-based binding assays use the natural fluorescence of tetracycline. Once the tetracycline−ykkCD complex forms, the fluorescence of tetracycline is quenched. The amount of quenching as a function of sensor concentration gives the binding affinity KDa measure of how well the ykkCD riboswitch recognizes tetracycline.

site-directed mutagenesis, plasmid preps, agarose and denaturing gel electrophoresis, restriction endonuclease digestions, enzyme kinetics, HLPC, RNA synthesis, and purification as well as fluorescent binding assays. This curriculum has been adopted for 5 years with approximately 150 students enrolled. The course was taught by three different instructors with backgrounds in biochemistry and/or molecular biology.

Because the ykkCD riboswitch regulates expression of a multidrug-resistance efflux pump, the curriculum touches an emerging problem in modern medicine: bacterial antibiotic resistance. According to the Center for Disease Control, 70% of bacterial strains are resistant to at least one antibiotic, making treatment of common bacterial infections increasingly difficult. The most studied resistant superbug, methicillin-resistant Staphylococcus aureus (MRSA), is responsible for 369,000 hospitalizations that result in 19,000 deaths per year, adding at least $4 billion to health-related spending each year in the United States alone.12 Thus, there is an increased urgency to understand how bacterial defense mechanisms against antibiotics are triggered and to raise awareness of this growing problem by incorporating it into the undergraduate biochemistry curriculum.

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NOVELTY OF THE CURRICULUM The instructors find that the curriculum has three major benefits: (i) its usage of RNA as model system; (ii) its extensive coverage of bioinformatics; and (iii) its effectiveness in combining expository and project-based laboratory experiments. Using an RNA molecule as model system in project-based undergraduate laboratory experiments has several advantages. First, experimental procedures used to synthesize and purify nucleic acids are practically the same regardless of sequence. The instructors choose to design mutants to the ykkCD riboswitch RNA because this RNA represents the research interest of the author, but any other biologically active RNA or DNA molecule can be chosen as target with minimal modification of the protocols. Second, nucleic acid studies fit well within the 3 h per lab time typically allocated for undergraduate lab courses. Third, as a result of a plethora of research conducted since the 1980s, RNA gained recognition as a major player in cellular processes with demonstrated enzymatic and regulatory activities.15 Hence, familiarity with techniques used to study biologically active RNAs is a must to young biochemists entering the job market. Another breakthrough of the last 20 years happened in the field of bioinformatics. Following this curriculum, students learn how to access genome databanks, perform sequence alignments,13 primer design, and nucleic acid secondary structure prediction16skills that serve them well in today’s competitive job market. A continued increase in the availability of data mining programs that help interpret the tremendous

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SUMMARY OF THE CURRICULUM The curriculum is made up by an expository module and a project-based module. The expository module contains five lab sessions that cover topics not included in the project-based module such as enzyme kinetics or HPLC. In the project-based module, students first identify the evolutionarily conserved regions of the ykkCD riboswitch using multiple sequence alignment of ykkCD riboswitch sequences from Gram-positive bacterial species.13 Then, students investigate how the ykkCD riboswitch recognizes the antibiotic tetracycline by designing point mutations in these evolutionarily conserved regions. Finally, students test the impact of this mutagenesis on tetracycline recognition by determining the binding affinity of the tetracycline mutant ykkCD riboswitch complexes (Figure 1B). The mutants are further analyzed by the author’s research group, hence tying undergraduate teaching to basic research. Table 1 provides a summary of goals, techniques, and objectives for the lab. During this 15-week lab sequence, students gain experience with a variety of techniques widely used in academic and research settings in biochemistry: bioinformatics, primer design, B

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Use molecular visualization software Chimera to observe structural features of enzymes Measure steady-state kinetic parameters, fit and interpret data Separate phosphatidylcholines using reverse-phase HPLC

Study small molecule binding to a protein using fluorescence Titration, determine pKa, buffer capacity

Isolation and UV spectrometry of linear DNA; transcription Purification of mutant RNA using spin columns; UV spectrometry and denaturing PAGE to assess RNA purity Fluorescence quenching-based binding assays; determination and interpretation of binding affinity (KD)

Reinforce that most biochemical reactions cause change in pH; hence, biological systems must be buffered to prevent tissue damage. Highlight the importance of visualizing protein secondary structure, active site location, protein charge distribution, and animations to understand the strategies enzymes use to catalyze reactions. Review steady-state kinetics; determine KM, Vmax, kcat to evaluate the effectiveness of acetylcholine esterase, one of the enzymes that achieve catalytic perfection. Learn about the structural properties of membrane lipids. Review principles of the important separation methods reverse-phase HPLC. Possible exam.

Contrast fluorimeter and fluorescent plate reader as tools to measure binding affinity.

Reinforce RNase free lab techniques; contrast agarose to polyacrylamide gel electrophoresis. Encourage discussion about the reasons for poor RNA yield and quality. Review the definition of KD value and fluorescence quenching. Highlight the inverse relationship between KD and binding affinity. Relate Krel D = KD(mutant)/KD(wild type) to the importance of mutated nucleotides in tetracycline recognition.

Design two mutants; one that changes a conserved secondary structure and one that restores the original structure (compensatory mutation). Discuss why the mutations caused the observed structural changes. Design primers for one mutant; relate primer GC content, Tm, and success of mutagenesis. Set up PCR; possible exam. Relate methods of quality control (agarose gel, UV spec.) to physical properties of DNA (negatively charged backbone, aromatic bases). Encourage discussion about reasons for low DNA yield and purity. Review theory behind specificity of restriction endonucleases and their use in life science research. Encourage discussion about reasons for inefficient digestion. Review mechanism of transcription; possible exam.

Appears simple, but necessary for project success. Design a mutant that changes conserved sequence element; encourage discussion about why they choose that mutant.

Learning Objectives and Comments

A detailed description of the experiments, materials, and equipment needs are found in the Supporting Information. See also ref 14. bThese are expository laboratory experiments.

HPLCb

Molecular visualization of enzymesb Enzyme kineticsb

Investigating buffersb

Determine binding affinity of mutant RNA−tetracycline complex Drug binding to serum albuminb

Mutant RNA purification

Review pipet and centrifuge usage and sterile techniques Introduction to gene data banks and sequence alignment programs Introduction to secondary structure prediction programs

Introduction Identify conserved sequence segments in RNA Predict secondary structure of RNA Design mutagenic primers Perform mutagenesis Purify plasmid DNA that carries the mutant RNA Perform restriction enzyme digestion of DNA Mutant RNA synthesis

Quickchange vs traditional mutagenesis Quickchange PCR Plasmid prep, UV spectrometry of DNA and agarose gel electrophoresis Restriction digest of DNA and agarose gel electrophoresis

Techniques/Methods

Goal for the Week

Table 1. Summary of Goals, Techniques, and Objectives for Each Laba

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Figure 2. Yield and purity of plasmids and RNA synthesized in the teaching lab are comparable to those produced in research lab settings. (A) Vector map of the puC19-ykkCD construct. Recognition sites for EcoRI and BamHI restriction enzymes are shown. T7 promoter was added to the ykkCD sequence to ensure efficient transcription with T7 RNA polymerase. (B) Agarose gel electrophoresis of puC19-ykkCD plasmid DNA shows that the plasmid prepared is mostly supercoiled. Typical yield (per student team) was 0.1 μg/μL DNA in 200 μL total volume. (C) Denaturing PAGE (urea-PAGE) of the ykkCD toxin sensor RNA shows that the RNA obtained is free of degradation products. The high molecular weight contaminant in the unpurified RNA is most likely the template linear DNA that is removed during purification.

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amount of data from genome sequences, 3D structures, and microarrays is expected. As a result, preparing 21st-century biochemists to use these programs is a requirement. The extensive focus on bioinformatics and interpretation of binding affinities also increases the technical literacy of the student, a goal much sought-after by the National Education Standards.17 Technology in the classroom motivates students to work cooperatively and use outside resources to complete their tasks.18 The combination of expository and project-based laboratory experiments provides students and instructors with flexibility and the benefits of both traditional and nontraditional styles. The expository laboratory experiments may be used as skillbuilder units to equip students with the technical know-how that is pertinent to tackle a biochemistry project. Alternatively, the expository laboratory experiments may be strategically inserted within the project-based sequence to allow time for primer synthesis or DNA sequencing. The project−based sequence is also remarkably flexible. Mutant design, mutant synthesis, and functional tests of mutants can be taught in the order that benefits student and faculty the most. So far, the instructors have taught the sequence as listed above; alternatively, mutants from a previous year can be synthesized and analyzed first and, based on the results of the analysis, new mutants can be designed.

COURSE AND LEARNING OUTCOME ASSESSMENT

Instructor Evaluation of How Well Students Mastered the Curriculum

As observed by three different instructors, students’ skills gradually improved as the semester progressed. Instructors were encouraged that even less strong students retained a keen interest in the progress of the project and a desire to succeed. These students often go through laboratory experiments on “autopilot”, not absorbing much of the material taught. The instructors feel that a real-life research project is definitely the right “hook” to engage those students in the learning process. By the end of the semester, students became independent researchers. They were able to complete complex experiments by following a written protocola skill required by industry for entry-level positions. Relying on observations of instructors to evaluate the effectiveness of a curriculum is undoubtedly subjective. Because students come into an advanced laboratory course with various skill sets and attempt to master complex experimental techniques, a basic skill test could not be used to measure their improvements. Comparison of Data Generated by Introductory Biochemistry Students to Data Quality Typically Observed in a Research Lab

On the basis of a five-year study, instructors involved in the course concluded that the experiments outlined were doable at the undergraduate level. DNA and RNA yields as well as purities were comparable with those obtained in a research lab (Figure 2). D

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Figure 3. Binding affinity of mutant ykkCD sensor RNA−tetracycline complexes was determined in the teaching lab with enough accuracy to serve as preliminary data for more extensive study in the research lab. Top panel from left to right: Secondary structure prediction of the wild-type and three student-designed mutants of the Bacillus subtilis ykkCD sensor RNA. Nucleotides with >90% sequence conservation are shown in red; nucleotides with 50−90% sequence conservation are shown in blue (the same as in Figure 1). Mutated nucleotides are boxed. Secondary structure predictions were generated by mfold. Bottom panel: Fluorescent quenching as the function of RNA concentration was used to determine binding affinities (Prizm). KD values listed represent averages (mean) and standard deviation of at least three independent measurements. More accurate error analysis is typically not used for KD values because the difference in KD has to be at least an order of magnitude to be considered significant. For example, a KD increase of an order of magnitude corresponds to about 1 kcal/mol in binding energies, which often means the loss of a H bond. Anything less than loss of one H bond between RNA and ligand is hard to interpret at the molecular level.

each mutant was prepared and analyzed by 2−3 student teams. This practice provided a built-in control and made the lab manageable. The top mutants were selected somewhat arbitrarily. First, mutations that had been done by a previous team were eliminated. Then, instructors gave preference to mutants that modified a conserved region of the RNA that had not been targeted before. In the future, if a 3D structure of this RNA becomes available, mutant selection could be guided by analysis of ykkCD-tetracycline crystal structures.

Initially, RNA yields were below expectations for about 20% of the students, thereby making measurement of binding affinity difficult. As a result, the instructors replaced the smallscale plasmid prep with a medium-scale plasmid prep that produced more DNA template for transcription, which in turn resulted in higher RNA yields. With this strategy, each student team had sufficient RNA to set up at least one binding assay and analyze the impact of their mutant on ykkCD riboswitch function (Figure 2, Figure 3). Binding isotherms were of acceptable quality, though accurate determination of KD values for weak binders was not possible due to the limited amount of RNA available. Standard deviations of KD values were higher than the gold standard of 20% or less; but this is expected considering the skill level of the experimenters (Figure 3).

Evaluation

Prelab quizzes were conducted before each lab to ensure that students came prepared to lab. Laboratory reports were turned in one week after each laboratory experiment to allow students to summarize and reflect on the experiments performed in lab. Three written examinations were conducted to evaluate how well students mastered the curriculum. These evaluation methods proved to be advantageous over evaluating students based on one or two reports because they differentiated between students better and allowed the instructor to give students frequent feedback on their performance. Frequent feedback helped students to improve the mastery of the curriculum as well as their problem solving and written communication skills.

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PRACTICAL SUGGESTIONS FOR INSTRUCTORS A detailed instructor’s manual is available in the Supporting Information. Mutant Selection

The ultimate goal of this lab course was to attract talented, enthusiastic students toward careers in science and equip them with skills essential to be successful in today’s tough job market. To make the curriculum manageable, each semester, the top 6 student-designed mutants were selected by the instructor for further analysis. Thus, in a lab course with 36 students enrolled,

Adoptability of the Curriculum

Instruments used by this lab sequence (PCR machine, fluorimeter, UV spectrophotometer, apparatus to perform gel E

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(3) (a) Murthy, P. P. N.; Thompson, M.; Hungwe, K. Development of a Semester-Long, Inquiry-Based Laboratory Course in Upper-Level Biochemistry and Molecular Biology. J. Chem. Educ. 2014, 91 (11), 1909−1917. (b) Caspers, M. L.; Roberts-Kirchhoff, E. S. An Undergraduate Biochemistry Laboratory Course with an Emphasis on a Research Experience. Biochem. Mol. Biol. Educ. 2003, 31 (5), 303−307. (c) Bailey, C. P. RNase One Gene Isolation, Expression, and Affinity Purification Models Research Experimental Progression and Culminates with Guided Inquiry-Based Experiments. Biochem. Mol. Biol. Educ. 2009, 37 (1), 44−48. (4) (a) Hall, M. L.; Guth, C. A.; Kohler, S. J.; Wolfson, A. J. Advanced instrumentation projects for first-year biochemistry laboratory. Biochem. Mol. Biol. Educ. 2003, 31 (2), 115−118. (b) Knutson, K.; Smith, J.; Wallert, M. A.; Provost, J. J. Bringing the Excitement and Motivation of Research to Students; Using Inquiry and Research-Based Learning in a Year-Long Biochemistry Laboratory PART I-GUIDED INQUIRY-PURIFICATION AND CHARACTERIZATION OF A FUSION PROTEIN: HISTIDINE TAG, MALATE DEHYDROGENASE, AND GREEN FLUORESCENT PROTEIN. Biochem. Mol. Biol. Educ. 2010, 38 (5), 317−323. (c) Walter, J. D.; Littlefield, P.; Delbecq, S.; Prody, G.; Spiegel, R. C. Expression, Purification, and Analysis of Unknown Translation Factors from Escherichia coli: A Synthesis Approach. Biochem. Mol. Biol. Educ. 2010, 38 (1), 17−22. (d) Gray, C.; Price, C. W.; Lee, C. T.; Dewald, A. H.; Cline, M. A.; McAnany, C. E.; Columbus, L.; Mura, C. Known structure, unknown function: An inquiry-based undergraduate biochemistry laboratory course. Biochem. Mol. Biol. Educ. 2015, 43 (4), 245−262. (e) Hall, M. L.; Vardar-Ulu, D. An Inquiry-Based Biochemistry Laboratory Structure Emphasizing Competency in the Scientific Process: A Guided Approach with an Electronic Notebook Format. Biochem. Mol. Biol. Educ. 2014, 42 (1), 58−67. (f) Farnham, K. R.; Dube, D. H. A Semester-Long Project-Oriented Biochemistry Laboratory Based on Helicobacter pylori Urease. Biochem. Mol. Biol. Educ. 2015, 43 (5), 333−340. (g) Garrett, T. A.; Osmundson, J.; Isaacson, M.; Herrera, J. Doing that thing that scientists do: A discovery-driven module on protein purification and characterization for the undergraduate biochemistry laboratory classroom. Biochem. Mol. Biol. Educ. 2015, 43 (3), 145−153. (h) Silverstein, T. P.; Kirk, S. R.; Meyer, S. C.; Holman, K. L. M. Myoglobin structure and function: A multiweek biochemistry laboratory project. Biochem. Mol. Biol. Educ. 2015, 43 (3), 181−188. (i) Knutson, K.; Smith, J.; Nichols, P.; Wallert, M. A.; Provost, J. J. Bringing the Excitement and Motivation of Research to Students; Using Inquiry and Research-based Learning in a Year-long Biochemistry Laboratory PART II-RESEARCH-BASED LABORATORY-A SEMESTER-LONG RESEARCH APPROACH USING MALATE DEHYDROGENASE AS A RESEARCH MODEL. Biochem. Mol. Biol. Educ. 2010, 38 (5), 324−329. (j) Willhite, D. G.; Wright, S. E. Detergent-Based Isolation of Yeast Membrane Rafts AN INQUIRY-BASED LABORATORY SERIES FOR THE UNDERGRADUATE CELL BIOLOGY OR BIOCHEMISTRY LAB. Biochem. Mol. Biol. Educ. 2009, 37 (6), 349−354. (5) (a) Dunne, C. R.; Cillo, A. R.; Glick, D. R.; John, K.; Johnson, C.; Kanwal, J.; Malik, B. T.; Mammano, K.; Petrovic, S.; Pfister, W.; Rascoe, A. S.; Schrom, D.; Shapiro, S.; Simkins, J. W.; Strauss, D.; Talai, R.; Tomtishen, J. P.; Vargas, J.; Veloz, T.; Vogler, T. O.; Clenshaw, M. E.; Gordon-Hamm, D. T.; Lee, K. L.; Marin, E. C. Structured Inquiry-Based Learning: Drosophila GAL4 Enhancer Trap Characterization in an Undergraduate Laboratory Course. PLoS Biol. 2014, 12 (12), e1002030. (b) Parra, O. a. P. A Research-Based Laboratory Course Designed To Strengthen the Research-Teaching Nexus. Biochem. Mol. Biol. Educ. 2010, 38 (3), 172−179. (c) Johanson, K. E.; Watt, T. J. Inquiry-Based Experiments for Large-Scale Introduction to PCR and Restriction Enzyme Digests. Biochem. Mol. Biol. Educ. 2015, 43 (6), 441−448. (d) Smith, J. T.; Harris, J. C.; Lopez, O. J.; Valverde, L.; Borchert, G. M. ″On the Job″ Learning: A Bioinformatics Course Incorporating Undergraduates in Actual Research Projects and Manuscript Submissions. Biochem. Mol. Biol. Educ. 2015, 43 (3), 154−161.

electrophoresis, gel documentation system) are found at most undergraduate institutions and would be used in biochemistry laboratory experiments regardless of curriculum. Nucleic acid sequence databanks and programs to manipulate these data as well as programs used in primer design are available free of charge; thus, teaching bioinformatics does not represent a significant financial burden to institutions.

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SUMMARY The ultimate goal of science educators is to recruit talented students into careers in science. Project-based biochemistry laboratory experiments are keys to deliver research opportunities to a large number of students, especially at smaller, teaching-oriented institutions or community colleges. Students, when given the opportunity to do real-life research, learn how the content presented in textbooks is generated. Getting the taste of real-life research underscores that science is a method to understand how the world works and not a collection of facts that never change. Despite all of these benefits, project-based laboratory experiments are often not sufficient to give students a good coverage of techniques used in academia or industry because they only cover techniques that are needed for the project. Moreover, students may not have the research skills necessary to tackle a research project on day one of a biochemistry lab course. The combination of expository and project-based laboratory experiments provides the “best of both worlds” experience to students and instructors.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.6b00578. Instructor manual (PDF; DOCX) Sample quizzes and exams (DOC) Experimental protocols (PDF; DOC)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Timea Gerczei: 0000-0002-3519-7056 Notes

The author declares no competing financial interest.

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ACKNOWLEDGMENTS Ball State University IBC authorized the recombinant DNA work under case 824350-1. The author thanks students enrolled in CHEM 465 biochemistry laboratory course at Ball State University, Muncie, IN for their excellent work and dedication and Anita Gnezda and Scott Pattison for teaching the course and critically reading the manuscript.

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