Bacterial Diversity Studies Using the 16S rRNA Gene Provide a Powerful Research-Based Curriculum for Molecular Biology Laboratory

Abstract

We have developed a ten-week curriculum for molecular biology that uses 16S ribosomal RNA genes to characterize and compare novel bacteria from hot spring communities in Yellowstone National Park. The 16S rRNA approach bypasses selective culture-based methods. Our molecular biology course offered the opportunity for students to learn broadly applicable methods while contributing to a long-term research project. Specifically, students isolated and characterized clones that contained novel 16S rRNA inserts using restriction enzyme, DNA sequencing, and computer-based phylogenetic methods. In both classes, students retrieved novel bacterial 16S rRNA genes, several of which were most similar to Green Nonsulfur bacterial isolates. During class, we evaluated student performance and mastery of skills and concepts using quizzes, formal lab notebooks, and a broad project assignment. For this report, we also assessed student performance alongside data quality and discussed the significance, our goal being to improve both research and teaching methods.

FIG. 1

Student-generated flow chart depicting large-scale plasmid isolation procedures, Unit 1. This component of the prelab assignment comprised 10% of each Unit Lab grade. Evaluation notes in upper right corner are instructors additions.

FIG. 2

Phylogenetic tree using representative class data. The tree was generated using maximum parsimony methods against a dataset of known bacteria (indicated in italics with accession number in parentheses). The bar indicates 10 nucleotide changes. Branch numbers indicate percent support for that branch. Bacterial lineages are indicated in brackets.

FIG. 3

Representative PCR data. Panel A was generated using general bacterial 16S primers. Panel B was generated with GNS-specific primers. Lane 1 in both A and B is marker standards (Lambda/HindIII), and lanes 2 through 12 were representative products using different PCR buffers. The arrows at the right indicate the target fragment. PCR product was separated using 1% agarose with standard TAE running buffer.