AN EVALUATION OF FUNGAL BIOLOGY TECHNIQUES IN ACADEMIC RESEARCH AND IN THE HIGH SCHOOL CLASSROOM

Abstract

Fungal gene replacement research This report summarizes how a diverse internship experience provided a working knowledge of the basic techniques required to use fungi in a laboratory setting, and evaluates the usefulness of translating those techniques into the high school classroom. To gain and demonstrate an understanding of fungal biology, a project was undertaken in Dr. Marc Orbach’s laboratory with the goal of creating a mutant strain of Magnaporthe oryzae lacking the putative virulence gene CPS1. An attempt was made to establish stable knockout mutants of M. oryzae strain Guy11 through Agrobacterium tumefaciens mediated transformation (ATMT) and protoplast transformation techniques (Betts, 2007). A gene encoding hygromycin B phosphotransferase (Hph) which provides resistance to hygromycin was used in both cases to attempt to replace the target M. oryzae CPS1 DNA. Putative transformants were analyzed by PCR and agarose gel electrophoresis to compare observations to expected results. Hygromycin resistant transformed strains were obtained using two different M. oryzae strains, Guy11 wild type and Guy11Δku80 mutant lacking the gene essential for Non-homologous end joining DNA recombination. Complementary to the PCR and electrophoresis analysis, Southern blot analysis was employed to examine the CPS1 locus in the putative transformants. Education outreach To demonstrate the effectiveness of fungal biology techniques in the classroom, an experiment was designed to convey Alexander Fleming’s discovery of penicillin. An activity was designed to communicate the variability of antibiotic sensitivity among bacterial species to high school students. Classroom resources mandate a simple experimental design to communicate scientific concepts to high school students. A protocol was developed and optimized to demonstrate the interaction between Penicillium chrysogenum and three different species of bacteria, using materials that are readily available to high school classrooms. Classrooms with student populations who are predominantly underrepresented in higher education and who are from economically depressed areas became the model for resource availability so that the protocol could be used by students from a wide range of backgrounds. For the classroom activity, Penicillium chrysogenum conidia were harvested from potato dextrose agar (PDA) media and quantified by optical density using a spectrophotometer before culture flasks of liquid media were inoculated. After several days of fungal growth, cultures of Staphylococcus epidermidis, Micrococcus luteus, and Enterobacter aerogenes were inoculated into separate cultures of P. chrysogenum as well as sterile liquid media in culture flasks. After 24 hrs the cultures were filtered to separate fungi from bacteria and the growth of the bacteria was quantified by optical density and compared across cultures. The classroom activity, titled “Penicillium Antibiotic Effect”, has been added to the BIOTECH Project website (http://biotech.bio5.org/biotech_lab_activities/penicillium) and an article was written and submitted to the American Biology Teacher journal of the National Association of Biology Teachers.