by Gen Nelson, Germantown Friends School, Philadelphia (www.gfsnet.org)
For six weeks during the summer of 2011, I worked with Dr. John Hall and Dr. Rick McCourt in the Laboratory for Molecular Systematics and Evolution at the Academy of Natural Sciences in Philadelphia. The work was supported by a Research Experiences for Teachers supplement to the Green Algal Tree of Life project (www.gratol.org). Although I have always considered myself to be an animal-oriented biologist, this experience gave me a new appreciation for the beauty and complexity of algae, as well as a better understanding of the light algae shed on evolution, and the ways in which algae can be incorporated into high school biology curricula.
Before I started teaching, I worked as a laboratory technician at the University of Pennsylvania School of Dental Medicine. Although I am comfortable in a laboratory setting, molecular biology techniques have advanced considerably since the mid-1980s, and I am grateful to Dr. Hall and Dr. McCourt for sharing their expertise with me. During the course of this internship, I learned a variety of techniques, including two protocols for extracting DNA from algae, algal cell culture, polymerase chain reaction, and microscopic algal identification. Dr. Hall and I also took several field trips, which afforded me opportunities to learn where to find algae and how to collect, store and process field samples.
My advanced biology students are direct beneficiaries of this internship, as I developed three activities over the summer that I have used in my classroom this year.
MEET THE ALGAE
The first activity, “Meet the Algae” introduces students to microscopic algal identification and the use of polymerase chain reaction (PCR) and agarose gel electrophoresis to distinguish morphologically similar species. Dr. Hall created two mixed cultures of algae (Mix A and Mix B), each of which contained six genera, including one filamentous genus, and a dichotomous key to accompany the mixtures. The filamentous genus in Mix A was morphologically quite similar to the filamentous genus in Mix B, so it was difficult for students to determine which of these two genera their sample contained. Dr. Hall came to my classroom to assist students in this work. After briefly explaining how to make a wet mount and how to use the key, students were challenged to describe, identify and sketch as many of the genera as they could.
Because microscopy was insufficient to distinguish between the two filamentous genera, students used a molecular technique, polymerase chain reaction (PCR). Dr. Hall provided us with monocultures of each of the filamentous algae and appropriate primers for the amplification of the large subunit of Ribulose bisphosphate carboxylase/oxygenase (rbcL). Based on known sequences of this gene, Dr. Hall designed primers that would amplify the gene in one filamentous species but not the other. This enabled students to identify which genus was in their mixture based on the results of their PCR–one amplification would yield a DNA fragment whereas the other would not, which provided a simple, DNA sequence based identification tool.
After completing their descriptions and sketches, students extracted DNA from the appropriate monoculture, set up a PCR reaction, and ran the resulting amplified DNA fragments on agarose gels. Aside from a few inevitable slips in technique (in themselves, these slips were teaching moments), this activity worked as we expected it to, and students successfully learned to observe and identify algae with a microscope, DNA extraction, PCR and electrophoresis. On a broader level, students learned that morphological examination is not always sufficient for identifying algae, and molecular techniques provide additional evidence that can be useful when morphological evidence is equivocal. They learned that DNA sequences are thus another part of taxonomic identification, an important lesson in modern systematics. This activity also provides practical experience with the applications of PCR and electrophoresis.
PHYLOGENETICS AND EVOLUTION
Evolution is a major theme of my advanced biology course, and during second semester my students work in small groups on research projects about phylogenetics. In order to prepare them for this work, I developed an activity about vascular plant ancestry that involves determining which of three large groups (clades) of algae are the closest relatives of vascular land plants (Tracheophyta). Students observe photographs of specimens from each of these algal groups: green algae, Chlorophyta; red algae, Rhodophyta; and brown algae, Phaeophyceae. Students and formulate preliminary hypotheses about which group is most similar to vascular land plants. They then use the nucleotide database at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/) to find and download rbcL sequences from 3 members of each of these groups. Using pairwise alignments, students construct a distance matrix that shows % identity scores for each pair of algae, and then use this distance matrix to determine which group of algae is most closely related to Tracheophytes. From this activity, students learn how to mine data out of large repositories of nuclear sequences, execute pairwise alignments and interpret what an alignment means, and determine evolutionary relationships based on sequence similarity.
BUILDING PHYLOGENETIC TREES
The third activity I developed during my summer in Dr. McCourt’s lab builds on this vascular plant ancestry lab, and involves using the sequences students found to produce phylogenetic trees using three different algorithms (BioNJ, parsimony and maximum likelihood). We use SeaView4, freeware that runs locally on our classroom laptops, to create multiple alignments and trees, and students analyze the topology, strengths and weaknesses of each of the trees they generate. They then attempt to improve the weakest parts of their BioNJ trees by using BLAST to find rbcL sequences from closely related genera or species, and add these sequences into their trees to see if increased taxon sampling improves the weak bootstrap support of some branches.. Aside from gaining familiarity with the way the software works and improving their data mining skills, this activity reveals the complexity of molecular phylogeny to my students. They are surprised to discover that different algorithms often result in different tree topologies, and that sometimes strengthening one part of a tree weakens other parts. On occasion, their trees disagree with their conclusions based on their distance matrices, and that leads to interesting discussions about the nature of truth and the meaning of terms like “degrees of confidence.”
TAKE HOME MESSAGE
In summary, I developed three specific lab explorations while working with Dr. McCourt and Dr. Hall, but the impact these experiences have had on my curriculum and my students goes well beyond these activities. Dr. McCourt and Dr. Hall have visited my classroom at least twice a year since my RET work, once early in the fall to help with the microscopy lab, and again later in the year to introduce “tree thinking.” These visits provide my students opportunities to have direct, personal interactions with practicing biologists, and this invariably enhances their understanding of what a career in the sciences entails. Through completing these activities, my students gain hands on experience in a number of molecular biology techniques, including DNA extraction, polymerase chain reaction and agarose gel electrophoresis. They also develop critical thinking and analytical skills through testing hypotheses and considering the strengths and weaknesses of multiple valid explanations. Finally, my students and I have gained a new appreciation of the beauty, diversity and complexity of algae.
Supported by Research Experience for Teachers Supplement to NSF DEB 1036478. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.