Basic DNA Methods Red Layer Microbial Observatory Using Microbial Diversity and Biotechnology to Enhance Learning

National Science Foundation Western Oregon University Yellowstone National Park

Introduction

Given that most RLMO bacteria cannot be grown in the lab, we study DNA isolated from these communities. Although some labs study whole genomes (sort of like the Human Genome Project), we describe small pieces of DNA – specifically, a segment called the 16S gene. This gene is studied and compared by MANY microbiologists. The 16S gene, found in all bacteria, contributes to the protein-building ribosome. DNA sequences from different 16S genes contain some regions that are similar and others that are not. In addition to telling us about who lives in RLMO communities, 16S sequences suggest environmental factors that may select certain organisms at different sites, and provide insight into the evolution and natural history of these communities.

Hypotheses Tested (1998-2000)

The main red layer bacterium is a new member of the Chloroflexi phylum – possibly an entirely new species.

These new Chloroflexi bacteria are more widely distributed in Yellowstone than previously thought.

Objectives (1998-2000)

(1)  To isolate and describe PCR-amplified 16S genes from Yellowstone RLMO communities

(2) To perform extensive surveys throughout Yellowstone for RLMO communities, comparing

(3) To archive and share these data via our on-line RLMO database

Key Data: The red bacteria were new members of the Chloroflexi phylum, most similar to a Japanese bacterium called Roseflexus, discovered in 2002. We were surprised at how diverse the red bacteria were, leading to several new hypotheses being tested. For a summary of this work, click HERE.

General Methods Description

Following sample collection, we extract genomic DNA by breaking open the bacteria using a bead-beating machine.  As with other kinds of samples, the DNA is then purified using organic solvents, alcohol, and salt.  Next, we use PCR (Polymerase Chain Reaction) to copy JUST the 16S genes.  We next have to clone the 16S genes in order to produce enough DNA for sequence analysis.  Cloning means pasting individual 16S genes into a mini-chromosome that can be grown in E. coli. Finally, we determine the DNA sequence of each 16S gene using our Li-Cor sequencer. Using a variety of software (e.g. BLAST, PAUP), we identify sequences and build evolutionary trees to study the relationships between many sequences. Important acknowledgements for bringing these methods to my lab go to: Brian Hedlund (UNLN), Bryan Dutton (WOU), and Danny Lodge (former research assistant, WOU).

Click here for technical information about:

Genomic Isolation and PCR	DNA Sequence Analysis – Reactions and Gels
Cloning and Working With Cultures	DNA Sequence Analysis – Reading and BLASTING
Plasmid Isolation – Screening and High Quality	Computational Biology Course (5 weeks)

Query the RLMO Database for Current DNA Sequence Information!