Curriculum

JOURNAL OF MICROBIOLOGY & BIOLOGY EDUCATION, December 2011, p. 194-199 Copyright © 2011 American Society for Microbiology DOI: 10.1128/jmbe.v12i2.327

Curriculum

An Investigation of Bacterial Protein Interactions as a Primary Research Project in a Sophomore-Level Molecular Biology Course † Jean A. Cardinale Division of Biology, Alfred University, Alfred, NY 14802 Longer term research activities that may be incorporated in undergraduate courses are a powerful tool for promoting student interest and learning, developing cognitive process skills, and allowing undergraduates to experience real research activities in which they may not otherwise have the opportunity to participate. The challenge to doing so in lower-level courses is that students may have not fully grasped the scientific concepts needed to undertake such research endeavors, and that they may be discouraged if activities are perceived to be too challenging. The paper describes how a bacterial protein:protein interaction detection system was adapted and incorporated into the laboratory component of a sophomore-level Molecular Cell Biology course. The project was designed to address multiple learning objectives connecting course content to the laboratory activities, as well as teach basic molecular biology laboratory skills and procedures in the context of a primary research activity. Pre- and posttesting and student surveys both suggest that the laboratory curriculum resulted in significant learning gains, as well as being well received and valued by the students.

INTRODUCTION The Molecular Cell Biology at Alfred University is a sophomore-level course taken by Biology majors and Biomedical Materials Engineering majors. The emphasis of the course is on cellular ultrastructure, organization, and function of cellular organelles, and on the regulation of selected cell activities. The course meets 3 hours a week for lecture, and 3 hours a week for lab. Prior to implementation of this laboratory curriculum, laboratory activities were fairly standard one- or two-week activities designed to reinforce theoretical concepts and provide hands-on experience with modern molecular cell biology methods such as PCR, electrophoresis, and restriction analysis. While students enjoyed lab activities, the course lacked any significant discovery or inquiry based activities. Additionally, students often failed to recognize the connections between common techniques and scientific concepts, or to understand how molecular biology techniques may be used within the greater context of a real-world research question. Therefore, the lab component of the course was transformed from the traditional layout to an experiential learning activity that more closely resembled how scientists work. The students in the course had the opportunity to participate in a novel research activity, which helped them to develop lab skills while also allowing them to make connections to a range of lecture concepts. Author’s mailing address: Division of Biology,Alfred University, One Saxon Drive, Alfred, NY 14802. Phone: 607-871-2205. Fax: 607-871-2359. E-mail: [email protected]. † Supplemental material available at http://jmbe.asm.org 194

The focus of the laboratory project was to gain insights into the function of a target protein, AniA, by detecting other proteins to which AniA might bind. For my students, the study of molecular biology is rather esoteric. The students have a hard time conceptualizing that cells and the biomolecules within are actually physical objects that interact with each other. The students also are challenged by the fact that molecular interactions happen in degrees — that there are weakly interacting or strongly interacting molecules, and that the function of molecules is influenced by this degree of interaction. For example, students sometimes have a hard time understanding that the strength of a promoter is based on how well transcriptional regulators or polymerases may bind the promoter. The Lex-based protein interaction system (2, 3, 4) provides a useful tool to address these conceptual challenges. The research project utilized the bacterial LexA-based two-hybrid protein interaction system (2, 3, 4). This system permits detection of protein homo- and heterodimerization in a haploid E. coli background, and allows for the identification of unknown fragments that interact with a target protein using a ‘bait and prey’ approach (Fig. 1). Daines et al. (2) designed expression plasmids so that the genes for target proteins could be cloned in frame with the gene for DNA binding domain of the LexA transcriptional regulator. The system also contains a reporter construct, which is expressed and detected when there is no interaction between target proteins, but repressed when target proteins interact. The strength of the interaction between target proteins may be visualized by the degree of development of purple color when transformants are grown on MacConkey plates. There are several advantages to using a bacterial protein-interaction detection system with students. Many molecular biology texts

Journal of Microbiology & Biology Education

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Cardinale: Bacterial Protein Interactions

also allowed us to link lab concepts to lectures on signal sequences and protein localization. In additional to reinforcing course concepts, students need opportunities to develop lab skills. This course was designed so that students had multiple opportunities to develop and hone lab skills such as pipetting and agarose gel analysis. Completion of the laboratory component of the course provided students with the opportunity to develop molecular biology skills that would allow independent research in subsequent years, and introduced microbiology to students who were unlikely to take future Microbiology courses. Intended audience Fig. 1. The LexA protein interaction detection system. General flowchart of the LexA protein interaction detection system, which allows students to identify putative proteins which interact with the gonococcal protein, AniA. A: Schematic of constructed plasmids. pANI contains the gene for the target protein in a LexDBDm low copy plasmid and is made and provided by the instructor. Students use a shotgun technique to generate a library of plasmids, each containing a section of the gonococcal genome in frame with the wild type LexDBD. B: Expression of chimeric proteins. Chimeric proteins containing LexDBD represent (in theory) all possible gonococcal proteins. C: If there is binding between AniA and a putative protein, the heterodimer binds to a chimeric operator (op+/opM) and prevents transcription of a lacZ reporter gene from the sulA promoter (PsulA)3. Colonies whose cloned proteins do not interact with AniA will express LacZ and appear purple on a MacConkey plate.

use the LacZ operon as an example of bacterial transcription regulation, and a direct connection between our reporter construct and this operon may be made. Cloning in bacteria as opposed to yeast excludes the need for a nuclear localization signal, and positive transformants may be detected sooner due to shorter generation times.The LexA-based system permits detection based on color change, and lends itself very well to conversations about strong versus weak protein interactions. AniA, the major anaerobically expressed protein of Neisseria gonorrhoeae, was chosen as the target protein. Students engineered expression plasmids potentially expressing fragments of all Neisserial proteins, and then determined if their expressed protein fragments interacted with AniA. AniA was selected as the target protein due to connections with lecture material. AniA is a copper-containing nitrite reductase with homology to electron transfer chain reductases. However, its location on the external surface of the outer membrane of Neisseria challenges the assumption that it is involved in energy generation (1). Use of this target protein permitted analogies to be made between the location of energy-generating machinery in the mitochondria and in the gram-negative bacterium, and to reinforce lessons in chemiosmotic coupling and the use of alternate electron acceptors. The use of an outer membrane-localized protein Volume 12, Number 2

This activity is most appropriate for Microbiology/ Biology majors or Biotechnology majors. Learning time This activity is designed to be the major project in a 15-week semester, in which each lab section meets for one 3-hour period per week. If students have prior micropipetting experience and sequencing is sent off-site, the activity may be completed in eight 3-hour periods. We required students to follow-up outside of lab times on days following transformant plating. Please see Instructor version for a more in depth discussion of modifications to the time line. Prerequisite student knowledge Students should have one semester of cell-based introductory biology and one semester of general chemistry. Topics typically covered in a cell-based intro biology course should include: Central Dogma, the processes of transcription and translation, and basic differences between eukaryotic and bacterial cells. Some knowledge of genetic engineering, such as restriction enzyme digestion, is helpful but not necessary. Pipetting skills are helpful, but not necessary as the first lab period of a 15-week semester may be dedicated to teaching these skills. Learning objectives Upon completion of this laboratory, students will be able to: • • • • •

Compare and contrast genes, plasmids, and genomes Explain the principle behind DNA prep kit protocols (alkaline lysis protocol) Describe what a protein:protein interaction is, and why it is important to identify protein:protein interactions Describe how a DNA binding protein is able to recognize a nucleotide sequence and bind DNA Explain the link between transcriptional repression


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• • • • • • • • •

and blocked protein expression for the correlating gene-product Explain the need for selection following transformation Explain the relationship between a restriction enzyme and a specific cutting site Outline the steps of basic genetic engineering experiment Outline the steps in the preparation of a shotgun clone library Use micropipettors with correct technique Pour, run, and analyze agarose gels Conduct a bacterial transformation experiment Perform restriction digestions Complete a novel molecular biology-based project, and communicate findings in written report format

PROCEDURE Materials Materials, equipment, and recipes are provided in Appendix 2. Student instructions Appendix 1 Student Manual, Parts 1–7 are provided to the students at the start of the semester. Part 8 Sequence Analysis is given to the students digitally at the beginning of the period in which sequence analysis is being completed. Instructor version This lab includes a series of activities that together allow students to complete a portion of a molecular cloning Table 1. Semester timeline. Week

Lab Activity

1

Podcast: Introduction to project

2

Introduction to the lab, lab safety, micropipetting

3

Part 1: Plasmid isolation

4

Part 2: Agarose gel electrophoresis

5

Part 3: Clone library construction

6

Part 4: Clone library isolation

7

Lab practical (micropipettors)

8

Mid-term break

9

Part 5: Double transformant construction

10

Part 6: PCR

11

Part 7: Sequencing reactions and gel

12

Part 7: Sequencing reactions and gel

13

Part 8: Sequence analysis

14

Lab practical (agarose gel)

15

Lab cleanup

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experiment over the course of a semester.The timeline of the semester is presented in Table 1.The activity reinforces major concepts covered in a sophomore-level Molecular Biology course, plus teaches common molecular biology techniques. These major concepts are listed below, and elaborated on in Appendix 2 (page 19, linking lab to course concepts.) • • • • • •

Central Dogma Chemiosmotic coupling (electron transport chain, energy generation) Transcriptional control (gene regulation, the lac operon) Protein function (protein interactions) Genetic engineering Metabolism (sugar usage, fermentation)

Given the length of the activity, specific instructor notes on each of the individual parts are given in Appendix 2. Information on strains and plasmids that make up the LexA protein interaction system (3) is also provided in Appendix 2. This activity can be challenging for sophomore-level students without guidance and support. It is necessary to engage the students on a weekly basis to assess their comprehension of the material. While formal examples of student assessment are given below, I make a habit of engaging each individual student several times throughout the lab period to make sure they are on track both with the protocol and with how their work fits into the big picture. I cannot emphasize how important this informal interaction is to their understanding of the big picture. At the beginning of the semester, many students are intimidated by the scope of the project and doubt their ability to comprehend it. My role is as much cheerleader or coach as it is instructor, and my belief that they can get it in the end is critical. The introductory PowerPoint presentations used to introduce the lab to the students are provided in Appendix 3. I have found that recording these presentations with audio (which may be done directly in PowerPoint or other lecture capturing software) and allowing students to digest them at their own pace leads to less panic in the beginning of the semester. Additionally, students will reference the presentations later in the semester, as needed. Suggestions for determining student learning I use a series of written assignments and practical exams to gauge student learning. The written assignments are provided in Appendix 4, along with rubrics and grading sheets. If students are not prepared for lab or show up late, short lab quizzes are given immediately as the lab period begins. There are two prelab assignments given during the semester. Prelab assignment #1 is included in the student manual. Prelab assignment #2, which has identical questions but focused on Part 6 PCR, is assigned orally a week before the Part 6 lab is to be done. Two lab periods are set aside during the semester for a lab practical, focusing first on micropipetting skills and later on agarose gel skills.


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A description of the practical set-up and expectations is provided in Appendix 4. Safety issues Safety glasses, lab coats, and gloves should be worn. Many molecular biology kits contain chemical hazards such as chaotropic reagents or reagents that are strong acids or bases. The manufacturer’s instructions should be read carefully to identify any hazards present. Gel imaging relies on UV light, and appropriate personal protective equipment should be used to prevent exposure to this light. The LexA system employs BSL 1 level organisms; however, aseptic technique and care should be taken to prevent growth of contaminants. Electroporation and electrophoresis involve high electrical currents. Equipment should be free of mechanical defects and in good working order, and only used in the presence of individuals who know how to use them properly. If sequencing gels are poured, acrylamide monomer is a potent neurotoxin. Acylamide solutions should be purchased rather than powder to reduce exposure to the monomer, and appropriate personal protective equipment should be worn.

DISCUSSION Field testing This activity has been the primary lab component for the sophomore-level Molecular Cell Biology class for three years. In general, students were successful obtaining scientific results for each step of the activity; percentages of successful students are presented in Table 2. In 2007, we did not include alkaline phosphatase in the Part 3 restriction enzyme digestion, so most results obtained were recircularizations of the initial plasmids. The one student who did obtain an insert in 2007 did not prepare his sequencing reactions correctly. We did see a low percentage (< 50%) of single transformants with the fall 2009 class; Table 2. Number of students with scientific results at each stage of the project a . Fall 2007

Spring Fall 2009 2009

Part 1: Plasmid isolation

21/21

17/18

24/24

Part 3: Single transformants

20/20b

17/17

10/23

detectedc

17/20

14/17

22/23

Part 6: Colony PCR

17/20

17/17

21/23

Part 7: DNA sequence

20/20

13/17

21/23

Part 8: Insert present

1/20

2/13

1/23

Part 5: Interactions

a

Results are presented as (successful students / total students). transformations yielded no resulting colonies; 20/20 obtained transformants following repetition of the transformation experiment. c Interactions recorded as either strong or moderate. b Initial

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however, this was likely due to aging/inactive ligase rather than student performance. Despite the low success rate at this stage for the fall 2009 group, there was a significant degree of success among the 10 students that did obtain transformants and, as a result, at the end of the semester no student was sequencing the same material. It is important to communicate to the students throughout the semester that, while they have their own individual ‘piece of the pie’, the experiment is only possible because the class as a whole is providing the level of genome coverage needed. It is helpful to use numbers about the quantity of potential genes in the genome versus the number of proteins with which AniA is likely to interact. In addition, students need reassurance that ANY result (negative or positive interactions) in the end can provide valuable information about AniA’s function. It is important to help the class understand that if the class as a whole identifies only one or two interacting clones, then we have been wildly successful and that everyone did contribute to that result. One of the students from the fall 2007 class elected to continue the project by further examining the two putative interacting proteins identified in spring 2009 as the subject of her senior thesis.After cloning of the full length genes and repeating the experiments, she identified a positive interaction between AniA and these putative proteins. This result was particularly encouraging and supports the premise that sophomore-level students can generate usable scientific data. Perhaps more important than generation of data is student perception of the activity and evidence of learning (which is addressed below). An end-of-semester survey queried student perceptions of the activity. Forty-five of 53 students identified the experience as valuable, and identified the following reasons for that value: Ability to make broader connections Techniques / skills learned Continuity and seeing end result Interesting / Exciting Related to real world applications Forty-four of 53 recommend repeating the lab. When asked what they liked best about the lab, the most common answers were the techniques and skills they walked away with (22 students), and the scientific relevance and real-world connections (14 students). Evidence of student learning The effectiveness of content learning was assessed through pre- and posttesting of scientific concepts (Table 3, Appendix 4) and authentic assessment of lab skills via lab practicals (Appendix 4). The pre- and posttest questions were designed as open-ended essay style questions directly addressing the content-based learning objectives. Tests were graded anonymously using the provided rubric (Appendix 4). Normalized learning gains for each student (Fig. 2), as well as comparison of raw pre- and posttest scores (Fig. 3),


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Cardinale: Bacterial Protein Interactions Table 3. Pre- and posttest questions. 1. What is a restriction enzyme, and what is one way it may be used? 2. Why is it important to study protein-protein interactions? 3. Describe what a DNA binding protein is and how it is able to bind DNA. 4. Describe how a DNA binding protein can act as a transcriptional repressor. 5. Compare and contrast gene, plasmid, and genome. 6. What is a shotgun clone library? 7. What species of bacteria is commonly used in molecular cloning experiments? 8. Briefly outline the steps of a transformation experiment. 9. What is negative selection and why is it important in molecular biology? 10. What is the alakaline lysis technique?

indicate that all students left the class gaining some degree of knowledge across all learning objectives, with some areas displaying a greater take-away than others. Students seemed to understand protein function best (questions 2 and 4). Their poor responses on several of the other questions may be due, in part, to poor test taking skills. For example, many students identified that restriction enzymes cut DNA, but failed to indicate that the splice site occurs at a specific base sequence. If I followed up with a question of where a restriction enzyme would cut, most would indicate at a specific base sequence. Many of the responses to question 8 (“briefly outline a transformation experiment”) included all steps of the semester, rather than just a simple transformation. This suggests that the students might not yet see that larger research projects are the sum of many smaller research activities. I was somewhat dismayed that learning gains on question 9 remained low, even in the third year this lab was performed. Despite a full semester of molecular biology and a discussion of negative selection related to growth on antibiotic containing plates, most students failed to identify how molecular biologists take advantage of negative selection. Despite the lowerthan-expected scores on many posttests, the students still made significant strides based on their knowledge entering the semester. More than 75% of students scored 10 or below on the pretest (weak to no understanding), yet improved their scores an average of 15 points (partial to good understanding). In terms of practical skills, students as a whole performed well in the lab practical (class averages of 92%, 93%, and 92%, respectively). Possible modifications I have made use of this lab activity for three years using AniA as the target protein, and students have identified a number of possible interacting proteins. A next step would be to use one of these identified proteins as the target protein for subsequent years. The system and experiment, therefore, has the potential to generate a significant amount 198

Fig. 2. Normalized learning gains. Pre- and posttests were graded anonymously.The rubric (provided in Appendix 4 reflected a maximum score of 4 for each question, with a score of 3 representing the expected understanding. Normalized learning gains ((Scorepost - Scorepre)/(4-Scorepre)) were calculated for each question for each individual student, and then averaged for each question (listed in Table 3). Class averages are indicated by semester (Fall ‘07, Spring ‘09, Fall ‘09).

Fig. 3. Raw pretest vs. raw posttest scores. Raw scores for pre- and posttests were plotted for individual students. Both tests had a maximum raw score of 40 points; however, a score of 30 represented expected understanding was achieved. The dashed line indicates where equivalent pre-and posttest scores would lie on the graph. The placement of points above this dashed line indicates that all students did improve their understanding of the concepts presented with the activity. Students in the fall ‘09 class tended to score lower on the pretest; however, posttest scores are evenly distributed over the three years.

of student-generated results over a long period of time, with each successive year generating new data. Given the nature of the protein interaction system, any target protein of interest may be cloned into the low copy plasmid, allowing students to ‘fish’ for proteins that might interact with it. The activity can, therefore, be tailored to line up with an instructor’s individual expertise with any bacterial species. A more targeted modification would be to directly clone into each of the plasmids instead of using the shotgun approach. For example, I have had students identify


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putative protein systems in Chitinophaga pinensis as part of a bioinformatics class. Students in my Molecular Cell course could design primers to specifically amplify and clone these proteins, followed by examining interactions to determine if the proteins identified do, in fact, interact in vivo. The system lends itself well to further testing of potentially interacting clones. If students initially construct their library of plasmids in SU101, they may test for homodimerization of their protein fragments by plating the initial clones on MacConkey. Additionally, instead on simply observing color changes on MacConkey plates for homo- or heterodimerization studies, students may measure beta-galactosidase activity of their transformants and further quantify the degree of protein interaction taking place via enzyme assays. Finally, there is a considerable amount of flexibility in the semester schedule depending on instructor goals and resources. The two lab periods set aside for lab practicals may be combined into one period, or eliminated altogether. If sequencing is sent off site, one to two additional periods may be freed up, depending on preparation of DNA for off-site services. If asking the students to attend to plates outside of class time is unrealistic, the time freed up above may be used to allow students to analyze plates. It is important, however, that images of MacConkey plates are taken within a proper time frame to allow visualization of the correct color development. Instructors may also opt to have students isolate and digest genomic material for shotgun cloning rather than providing digested genomic DNA. For nonmicrobiology students, this genomic preparation should only be completed if the organism of interest is nonpathogenic. Finally, more advanced students may be challenged to select restriction enzymes for cloning and to play a more significant role in designing the DNA constructs.

SUPPLEMENTAL MATERIALS Appendix 1: Student lab manual Appendix 2: Instructor support material

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Appendix 3: Introductory PowerPoint presentation Appendix 4: Assignments and rubrics Appendix 5: Examples of student work Appendix 6: Sequence images and chromatograms

ACKNOWLEDGMENTS All reagents and general lab supplies were provided by the Division of Biology, College of Liberal Arts and Sciences, Alfred University. The DNA sequencer was awarded through the LiCor Genomic Education Matching Funds Grant, with matching funds provided by the Family and Estate of Dr. Elias ‘36 and Rosalind Bernstein ‘39 Fass. I am indebted to the community of educators that attend ASM’s Conference for Undergraduate Educators, who provided a knowledgeable and important sounding board via discussions about this activity as it was developed over the years. The author declares that there are no conflicts of interest.

REFERENCES 1. Cardinale, J. A. 2000. Structural and functional analysis of AniA, the major anaerobically induced outer membrane protein of Neisseria gonorrhoeae [PhD Thesis]. University of Rochester, Rochester, NY. 2. Daines, D. A., and R. P. Silver. 2000. Evidence for multimerization of neu proteins involved in polysialic acid synthesis in Escherichia coli K1 using improved LexA-based vectors. J. Bact. 182:5267–5270. 3. Daines, D. A., M. Granger-Schnarr, M. Dimitrova, and R. P. Silver. 2002. Use of LexA-based system to identify protein-protein interactions in vivo. Methods in Enzymology 358:153–161. 4. Dmitrova, M., G. Younes-Cauet, P. Oertel-Buchheit, D. Porte, M. Schnarr, and M. Granger-Schnarr. 1998. A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli. Mol. Gen. Genet. 257:205–212.


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