To use the basic tools and techniques of traditional and molecular microbiology
To investigate the diversity and identity of soil microorganisms in a sampling site of your own choosing
To make careful, unbiased observations and to record and analyze them for meaning and importance
To design controlled experiments and collect data from those experiments to answer questions that arise from your observations
To show data in effective figures or tables
To make and articulate conclusions from experimental results
To write intelligibly in scientific research report format about your investigation and its conclusions, including its significance or implications
Introduction to the Project
In the 1980's scientists discovered that, despite the unicellular character of microorganisms and their invisibility to us, the microbial world is much more diverse and numerous than the macroscopic world of plants and animals. We now know that microbial communities are as complex and interdependent as those of visible forms of life. Traditional measures of diversity relied on physical traits and assessment of microbial metabolism was used for microbial identification rather than for understanding community structure and organization. The use of physical traits in determing relationships between organisms has two major problems when it comes to microbes: 1) microbes are not as morphologically diverse as metazoans and their morphological traits are not indicative of evolutionary relationships and 2) there are no morphological traits common to both macroorganisms and microbes. In the 1980's Carl Woese suggested that the deoxyribonucleic acid (DNA) sequences of certain common genes could be used to measure relatedness among radically different organisms. He picked the genes that encode ribosomal RNA (rRNA). Ribosomes, the protein-RNA complexes that are the scaffold on which proteins are synthesized, are common to all cells, both prokaryotic and eukaryotic. Despite differences in size, the sequences of rRNA molecules contain regions that are highly conserved, thus highly similar. Woese chose the intermediate sized rRNA molecule, 16S rRNA in prokaryotes and 18S rRNA in eukaryotes because it was large enough to contain enough information for genetic comparisons but small enough for the gene to be sequenced easily.
Comparing sequences of the gene (16S DNA) that encodes 16S rRNA in different bacteria can be used to identify them with much greater accuracy than traditional identification tools that used a battery of metabolic and physical tests on cultured community members. It is also common now to use rDNA sequencing to deduce phylogenetic relationships between different bacteria and among organisms as diverse as bacteria and humans. Woese's ground-breaking work altered the tree of life and showed that the prokaryotic world was evolutionarily much older than expected and much more important.
Such advances in molecular tools for gene sequencing and in other types of microorganism identification dramatically expanded our knowledge of the contribution of microbes in their (and our) environment. It is estimated that 99.9% of microbes are "unculturable" - that is, currently not cultured by traditional methods leading to the so-called "Great Plate Count Anomaly"- that many more bacteria are present than appear as colonies on agar plates. Culture-independent estimates of the number of bacteria in a gram of soil are 109 - this is several hundred to 9,000 orders of magnitude greater than the number derived from culture-dependent methods. It has been speculated that there might be 10 billion species of bacteria on Earth!
Your goal in this semester long project is to use both culture-based and culture-independent molecular methods of bacterial identification and characterization to investigate the diversity of bacteria in soil from a Wellesley greenhouse habitat and to explore how this group of bacteria function as a community.
Molecular Strategy for a Culture Independent Approach to Identifying Bacteria from a Soil Sample
Isolate Genomic DNA from Environmental Samples or Cultured Isolates
PCR Amplify rRNA gene sequences from Genomic DNA from "Universal" Bacterial Primers
Clone 16S rDNA PCR products into a plasmid vector and transform E. coli with plasmid
Pick transformed E.coli colonies containing soil community 16S rDNA inserts; culture the bacteria overnight in 96 well blocks; create glycerol stocks in 96 well plates to freeze and send away for 16s rRNA gene sequencing
Analyze Diversity of Soil Bacterial Community & Infer Phylogenetic and Functional Relationships
Strategy for Identifying and Characterizing Bacteria by Culture Dependent Techniques
In Pairs- Select Soil Sampling Site in Greenhouse
Collect Soil Sample
Use Selective & Enrichment Tools to Obtain Pure Cultures of
Bacteria of Specific Groups
Lyse Cells & Amplify 16s rRNA genes in
bacterial isolates by pcr for identification by
gene sequence analysis using the
Ribsomal Data Base
Characterize bacterial isolates using
traditional physical, metabolic, and functional
We would like to thank Charles Deutsch, Patricia M. Steubing; Stephen C. Wagner and Robert S. Stewart, Jr.; Kyle Seifert, Amy Fenster, Judith A. Dilts, and Louise Temple; and the instructors of the Microbial Diversity Course at the Marine Biological Lab in Woods Hole, MA for their valuable assistance in the development of these labs.