Using DNA Technology To Explore Marine Bacterial Diversity in a Coastal Georgia Salt Marsh

Abstract

An important aspect of teaching biology is to expose students to the concept of biodiversity. For this purpose, bacteria are excellent examples. Prokaryotes were the first inhabitants on Earth, surviving and even thriving under very harsh conditions as new species continuously evolved. In fact, it is believed that there are more than 5 x 1030 prokaryotes living on Earth today (Whitman et al., 1998). Our current knowledge of these tiny organisms is very limited, and less than 1% of all bacterial species have been described (Horner-Devine et al., 2004). However, the prominent roles bacteria play in nature are not easy to overlook: Their functions range from providing essential nutrients to plants through nitrogen-fixation (such as for Rhizobium leguminosarum) to enhancement of nutrient absorption in animal intestines (such as for Escherichia coli). As a result, identifying unknown species of bacteria and extending our understanding of known ones are important tasks for 21st Century scientists. Individual bacteria are too small to be seen or distinguished by the naked eye, or even by a microscope, making identification difficult. The traditional ways to identify bacterial species are based on their morphological, developmental, and nutritional characteristics, but these may be both inefficient and inaccurate. Microbiologists now take advantage of the rapid development in DNA technology, using the sequence of the small subunit ribosomal gene, or 16S rRNA gene, as a type of molecular fingerprint to classify bacteria (Johnson, 1984). The advanced placement (AP) biology class at Cedar Shoals High School in Athens, Georgia, learned how to explore bacterial biodiversity using molecular fingerprinting. We collected marine water samples, isolated bacterial colonies, extracted DNA, amplified and sequenced the 16S rRNA genes, and then compared the sequences to an Internet database to reveal the identity of the isolates. The project began with a field trip to the salt marshes on Sapelo Island, a barrier island in coastal Georgia, and was completed at our high school and in a laboratory at the University of Georgia. Here we describe how the bacterial biodiversity exercise was carried out, and discuss options for source material for bacterial isolation and flexibility in scheduling the laboratory exercise modules. This laboratory has both educational and practical value for the students; it is appropriate for both AP biology and introductory college biology classes. Methods The laboratory is divided into three modules. In the first, basic ecology and microbiology techniques are used to collect a water sample from the environment and isolate bacterial colonies on solid growth medium in Petri dishes. In the second, molecular biology methods are used to extract genomic DNA from selected colonies, and amplify and sequence the 16S rRNA gene. In the third, bioinformatic tools are used to compare the sequences to those available in a national DNA database to obtain information about the identity and ecology of the isolates. Our class of 20 students was divided into groups of four. Each student isolated his/her own bacterial colony so that each group worked with four isolates (although not all were successfully taken through to sequencing). Class size for this exercise is flexible, but 24 or fewer students is recommended. The entire exercise takes about one month to complete, but time can elapse between the scheduling of each module. Phase One: Isolating Bacterial Colonies In our class, a seawater sample from a salt marsh on Sapelo Island, Georgia, was used for bacterial isolation. However, water, sediment, or soil samples can be obtained from virtually any natural environment as starting material for this exercise. Collecting Water & Isolating Bacteria Materials * sterile collection bottle * thermometer * salinity meter (refractometer) * 10 test tubes filled with 9 ml of sterile seawater for serial dilution * 10 plastic Petri dishes filled with YTSS agar or other appropriate medium (To make YTSS agar, mix 4 g yeast extract, 2. …