Molecular Evolution NSF-ILI
The purpose of this project was to introduce a new course in the undergraduate curriculum that would provide integrated training in the theory and practice of molecular evolution to the biology majors at the University of Richmond.
The course is entitled "Molecular Evolution" (BIO 325) and has a strong "hands-on" approach. The critical formula was to have small size classes to effectively engage student in the everyday techniques and problems faced in a molecular laboratory. Students are paired up in research teams to carry on experiments using a set of molecular techniques. Students isolated DNA from amphibian tissues. Subsequently, they cleaved the DNA using restriction endonucleases under different conditions, built subgenomic libraries in phage lambda Zap II, screened those libraries for 28S ribosomal DNA (rDNA) clones, and learned dideoxy chain termination sequencing by using the cloned rDNA.
Students liked the new course. Several students mentioned that they enjoyed learning and actually carrying out the molecular techniques themselves instead of being presented with demonstrations of techniques. In particular, students liked learning about the practical applications of the molecular techniques that they were learning and how to interpret DNA data and DNA analyses.
This project was designed to enhance the "hands-on" training and experience in molecular techniques for Biology majors at the University of Richmond. This was accomplished through a change from presenting students with the uses and advantages of molecular techniques (the approach used in other courses, e.g. genetics, cell biology) to teaching a new one-semester course called Molecular Evolution, in which students learn how to select, use, and carry out experiments with molecular techniques-ranging from restriction analysis to gene cloning and DNA sequencing- and instrumentation. The course has been taught twice already, and each time the class size has been limited to 12 students.
The goal was for students to complete the course with an understanding of the basic principles of evolution at the molecular level coupled with the "know-how"; of molecular technique. The latter will help them in a wide range of research-oriented careers, ranging from all the basic areas of biology through ecological, agricultural, and applied medical research, to the pharmaceutical and industrial world.
Students teamed up in pairs to carry out the collaborative research techniques. Each pair chose an amphibian species from the frozen tissues kept in the PI's laboratory. Each team was required to isolate genomic DNA, and some of the isolated DNA was kept to build genomic libraries. Each student pair was required to cleave part of the isolated DNA with restriction endonucleases under at least 3 different experimental conditions (e.g., different temperature, salt concentration, enzyme concentration, etc.). Cleaved DNA samples were run in agarose mini gels. Results were compared and discussed among all students pairs to learn the different outcomes and to understand the origin of possible problems that one may run into when DNA digestions are not successful. In this first part of the course, students also learned to use sterile techniques, sterilization procedures, agarose gel preparation and basic horizontal electrophoresis principles.
Subsequently, student pairs were given frozen stocks of 28S rDNA that had been previously cloned into plasmid DNA in E. coli cells (XL1-Blue strain). Each student pair was expected to prepare liquid media to culture the stock cells. Students were required to prepare one flask of media following the instructions and two additional flasks with modified media (e.g., lacking tryptone extract, lacking glucose, increasing 10X the amount of glucose, varying culture temperature, with no shaking of the culture flask, etc.). Results were compared and discussed among all groups. Students pairs then proceeded to learn to isolate plasmid DNA with the successful cultures. Students were expected to carry out mini- and maxi-plasmid isolation procedures. This served to introduce students to the use, function, and principles of supraspeed and ultraspeed centrifugation as well as spectrophotometry. At the same time, some of the cultures were used by students to learn plating techniques (including preparing media for plates, pouring plates, and streaking liquid cultures on solid media).
The isolated plasmid DNA was subsequently cleaved, using the techniques learned earlier to separate the cloned DNA from the plasmid DNA. The reactions were run in agarose gels to check for digestion results and size of cloned DNA piece. This gave the students the opportunity to purify DNA from agarose gels. The cloned 28S rDNA segment was purified from the agarose gel and kept for future use as probe DNA.
In the second part of the course, each student pair was required to build a genomic library using the isolated DNA and the basic techniques they had learned up to that point. DNA was cloned into Lambda ZAP II bacteriophage using a commercial kit. Different concentrations of the bacteriophages libraries were mixed with fresh XL1-Blue cells, plated and incubated at 37 degrees until plaques appeared. This component introduced students to the principles and mechanisms of lytic and lysogenic viral cycles. Each plate was lifted using nitran membranes and each membrane blot was denatured, neutralized, and dried to use subsequently in southern blotting.P> The previously isolated 28S rDNA segment was then used to create a radioactive label probe to screen the library blots. The probe was built using a commercial nick-translation kit. This introduced the students to nick translation principles and radioactive safety methods and procedures. The labeled 28S rDNA probe was used to screen the library blots by southern blotting. Students learned southern blotting and the principles and concepts behind this technique (e.g., DNA homology, DNA melting temperature, stringency conditions, false positive plaques). Subsequently, positive plaques were isolated from the original plates and the 28S clone was excised from the bacteriophage and subcloned into plasmid DNA. Finally, the new cloned 28S rDNA was isolated and used to teach the student DNA sequencing and vertical electrophoresis.
Although the laboratory component of the course focused on techniques, this was not just a course in techniques. In parallel with learning skills in the laboratory, in the lecture component students also learned about mechanism of DNA evolution, methods of study and analyses of DNA, and applications of DNA data to address evolutionary questions. During the semester each student was required to analyze and present a research paper that used molecular techniques. Also, throughout the semester each student worked on writing a research proposal in the area of molecular evolution. The last week of the course was devoted to a symposium in which each student presented her/his research proposal in a poster format.
Students work was assessed in several ways. First, the course had a midterm exam and a final exam. Exams consisted of a mixture of factual and interpretative questions and problems to solve (e.g., population genetics, optimal sequence alignment, etc.). In addition to these exams, students were also graded on their oral presentation and on their research proposal. The grading of the research proposal was divided into two components, the research proposal itself (i.e., writing, clarity, ideas, feasibility of project, etc.) and the poster presentation of the research proposal during the final week of the semester (i.e., explanation of research proposal, clarity of figures, overall presentation of poster, etc.).
Also, students were required to keep a notebook of the laboratory procedures; these notebooks were randomly checked six times during the semester to verify that notes were properly kept.
Each of these activities accounted for one-sixth of the final course grade. The equal weight of all activities was design to merge together formal lecture activities with the laboratory component and students individual work, giving equal importance to each component.
The course was designed to give students their first hands-on experiences on molecular techniques and molecular evolution. The course was evaluated by the students. Students enjoyed this approach and several consistently indicated that they looked forward to coming to class and to the results of the experimental techniques they were learning in the laboratory. The students who enrolled in the course represented a variety of interests. A few wanted to pursue graduate school in biology, some wanted to go to medical school, and most were interested in allied health professions. All indicated that the techniques learned in the course would help them in their future long-term plans and in more short-term plans (e.g., searching for permanent jobs or for part-time jobs as laboratory technicians) while pursuing further studies.
Overall the course was well-received and liked by the students. It has served its original goal of engaging students in molecular biology and developing their interest in molecular evolution and biology. Two examples serve to illustrate this in particular. Ms. Leigh Merski and Mr. Shay Pratt enrolled in the molecular evolution course and by the end of the semester they were interested in continuing to work and learn more about DNA sequencing. As a result, Mr. Pratt wrote and submitted a proposal to the NSF-SURE (Summer Undergraduate Research Experience) program and received one of the 12 awards given for the summer of 1995. Mr. Pratt graduated in May, 1996 and his research experience helped him to secure a position working with the Institute of Genomic Research in Washington D.C. Ms. Merski continued the work she initiated in the molecular evolution course by taking research credit hours throughout the year. She also graduated in May, 1996 and is currently enrolled in the Bioengineering graduate program at Rutgers University.
A weakness of the course is that in order to carry out all the steps of the different molecular techniques, laboratory hours varied significantly from the assigned laboratory times. Students needed to come throughout the week and at different times of the day to check a specific step or particular result. Some students found that this conflicted with other requirements of their schedule. However, those same students recognized that it would be impossible to fit all experiments into a three-hour laboratory, and that they liked the flexibility of doing some of the experiments on their own time.
The course has been successful. The second year taught, students also learned the principles and techniques of Polymerase Chain Reaction (PCR) methods. In the future, PCR will be a regular component of course. Also next time I will teach this course, I plan to develop a manual that will go along with the laboratory component of this course.
The first time the course was taught, the course design was somewhat problematic because manual sequencing is largely time consuming and there was not enough time during the semester to collect sufficient data to analyze and compare with genetic data banks. The introduction of PCR methods the second time the course was taught helped to alleviate this situation. The incorporation of automated sequencing into the course will solve the situation completely.
Originally, the PI was planning to submit an article on teaching molecular evolution at the undergraduate level to the Journal of College Science Teaching. However, publication of articles in this journal is backed up for two years. Consequently, the PI decided to create a World Wide Web page of the Biology Department at the University of Richmond, that includes the present report (see attached printed copy). This page will be updated and modified to incorporate new information and methods as the course is repeated in future years. For example, the PI plans to incorporate the laboratory manual into this www page.
Last modified January 9, 1998.
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