(Difference between revisions)
Revision as of 20:39, 17 December 2009
Project: Soil Microbial Communities & Diversity
In this series of nine labs you will learn:
- To think, work, and write as microbiologists
- To use the basic tools and techniques of traditional and molecular microbiology
- To investigate the diversity and identity of soil microorganisms in a habitat 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 microbes invisibility to us, the microbial world is as, or more, diverse than the macroscopic world of plants and animals. Traditional measures of diversity relied on physical traits, but such criteria can not be used to assess relationships between microorganisms and macroorganisms because there are so few physical traits common to both. 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. We can also use rDNA sequencing to deduce relationships between different bacteria and among organisms as diverse as bacteria and humans. Woese's ground-breaking work altered the phylogenetic tree of life and showed that the prokaryotic world was evolutionarily much older than expected and much more important.
Recent 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 less than 1% of bacterial species have been described, primarily because so many are unculturable by traditional methods. Current culture-independent estimates of the number of bacteria in a gram of soil range from several hundred to close 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 and molecular methods of bacterial indentication to investigate the diversity of bacteria in soil from a Wellesley greenhouse habitat and to explore the specific role of some of the bacteria in that community.
Molecular Strategy for Indentification of Culturable & Non-culturable Bacteria Present in a Soil Sample
Isolate Genomic DNA from Environmental Samples
PCR Amplify 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 and transfer to 96 well plates for sequencing
Links to Labs in the Soil Microbes Project
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.