This kit enables a class of up to 28 students to isolate metagenomic DNA from environmental samples through PCR using primers of your choice, and analyze your results using agarose gel electrophoresis. Your class designs the experiment, desides on the focal group of the organisms and carries out the experiment.
|Metagenomics DNA Analysis Kit||reagents for metagenomic DNA isolation from 7 samples, PCR reactions, & gel electrophoresis $140 + S/H|
Who Lives There?
As you walk along a stream, you notice an orange plume coming out of the rocky bank into the stream. "Is that chemical natural or man-made" is probably the first thing you ask yourself. However, you may well want to ask "who lives there" because chances are there are lots of microbes present and their activities may be involved in the formation of the orange plume. Microbes are everywhere, both prokaryotic (DNA not organized in a nucleus) and eukaryotic (DNA organized in a nucleus), but how do we figure out who they are? That is the question this research kit helps you and your students to answer.
Microbial members of all 3 domains of life, Archaea, Bacteria, and Eucarya, live in all kinds of recognizable habitats such as oceans, lakes, and soil, but also in strange places like glaciers, hot springs, deep sea hydrothermal vents, and on and inside other organisms (DeLong, 1998; Pikuta et al., 2007; Rappe & Giovannoni, 2003). They can be found at temperatures from 0oC to over 100oC, at pH’s from < 1 to>10, at great pressures, at salt concentrations so high the salt starts to fall out of solution, and across a broad range of nutrient concentrations. Yet, we are still left with our question of how do we know who they are and this seemingly simple question is crucial to ecology.
Ecologists ask questions about individual organisms as well as populations, how they interact with their environment and with other organisms. Central to answering many ecological questions is the ability to count or estimate the number of individuals of a given species. If we are talking elephants or mature white oak trees, then it may not be that hard a task. If we have to determine the number of blue jays or spring peeper frogs, it won’t be as easy and our best bet is an estimate based on sampling. Yet, what if we are interested in microbes? For some, such as diatoms or ciliate protozoans, we could look at samples under the microscope although this will take some background knowledge of how to distinguish various taxa based on morphology. For bacteria, the choice for decades has been plating samples out on various culture media and counting colonies based on the assumption that each colony came from a single cell that was laid down at that spot. Colonies can then be identified using a variety of biochemical assays, stains, and growth-based tests. However, evidence has accumulated during the last two decades that cultured-based identification methods are not good estimators of microbial diversity because they catch only a small percentage (0.5-5%) of the microbial diversity present in any habitat at any time (Staley & Konopka, 1985). Even after a century plus of efforts, we only can get a minority of bacteria to grow in culture (Kaeberlein, Lewis, & Epstein, 2002). Given this problem, how can one detect the presence of a microorganism without growing it? Microscopy is possible, but it cannot separate live from dead cells nor can it identify strains to any fine degree by itself. However, microscopy using fluorescently-labeled DNA probes can identify strains to varying degrees based on sequence homology (DeLong, Wickham, & Pace, 1989). A genetics tool comes to the rescue! We typically think of genetics as just a set of concepts (e.,g., gene, chromosome) and processes (e.,g., meiosis, recombination), but genetics is also an experimental toolbox that can be used to address questions throughout the life sciences. Those questions may deal with medicine, agriculture, development, behavior, and ecology, yes ecology.
Another tool in our genetics toolbox is the technique Polymerase Chain Reaction or PCR (Sakai et al., 1985). PCR allows us to amplify specific sequences. The specificity of recognition in PCR comes from the choice of PCR primers that are used to start new DNA synthesis in each PCR cycle. The primers might be specific to a particular species, a genus, a family, or some higher level taxon (e.g., Blackwood, Oaks, & Buyer, 2005). Any gene is a potential PCR target, it just depends on the question you are addressing.
Research in Action in Your Classroom
Let us imagine for the moment that you are interested in the bacterial genus Salmonella which is in the news more often these days due to illness outbreaks usually linked to contaminated food supplies. You want to know if store bought chicken products carry Salmonella, but how can you answer that question? PCR could answer the question for if you had a DNA sample to test. Since you may not be able to grow Salmonella easily in your lab (or may not want to for safety reasons), it would be much easier to look for Salmonella-specific DNA sequences directly. As an alternative to culturing, you can swab the chicken products to collect any microbes present and isolate total DNA immediately. When one isolates DNA from all the microbes present in an environmental sample, this is called a metagenomic DNA sample because it represents all the microbes and their genomes (all their DNA). We can then carry out PCR on the metagenomic DNA sample using the Salmonella-specific PCR primers. Finally, we can detect the PCR products using agarose gel electrophoresis, a standard DNA lab technique.
Starting Ideas for Projects
Our initial line of PCR primers include those for E. coli, Salmonella, the domain Archaea, methanogenic members of the Archaea, and halophilic members of the Archaea. Using one of these primer sets, you could ask one of following questions but really you are only limited by your own curiosity.
We do have many other PCR primers available through our customization process, so let your curiosity soar.
References for More Information
Blackwood, Oaks, & Buyer, 2005. Applied Environ. Micro. 71:6193–8. Phylum- and class-specific PCR primers for general microbial community analysis.
DeLong, Wickham, & Pace, 1989. Science 243:1360-3. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells.
DeLong, 1998. Everything in moderation: Archaea as 'non-extremophiles'. Current Opinion in Genetics & Development 8:649-54.
Pikuta, Hoover, & Tang, 2007. Microbial extremophiles at the limits of life. Critical Reviews in Microbiology 33:183-209.
Kaeberlein, Lewis, & Epstein, 2002. Science 296:1127–9. Isolating "uncultivable" microorganisms in pure culture in a simulated natural environment.
Rappe & Giovannoni, 2003. Annual Rev. Micro. 57: 369-94. The uncultured microbial majority.
Saiki et al., 1985. Science 230:1350-4. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.
Staley & Konopka, 1985. Annual Rev. Micro. 39: 321-46. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats.