11664120
Description
| Question | Answer |
| Winogradsky column | artificial environment used as a long term source of diverse microbes for enrichment cultures: CaCO3 (buffer) and CaSO4 (sulfate source) at bottom, water from environment at top (for aquatic photosynthesizers) different functional groups will grow at different layers!! |
| Developing enrichment cultures | use select nutrient sources in media that (mostly) only target organism can use --> add sample to medium --> target organism multiplies --> plate onto agar medium, purify by isolating a colony of organism of interest (plating might be repeated a few times before doing this) |
| Results of enrichment cultures | Positive result: an organism that can use the nutrients in medium/simply survive is present, but we don't know by how much Negative result: inconclusive, no proof that a certain metabolism DOESN'T exist |
| enrichment bias | in liquid enrichment cultures, most dominant organisms are the ones that grow most rapidly EVEN IF they are not most abundant in actual environment |
| reasons for enrichment bias | 1) The enrichment culture is not an exact replica of the sample's environment 2) Nutrient concentrations are much higher in the lab 3) Types/proportions of other organisms are different in the lab 4) Temp, humidity, and chemical conditions are different in the lab |
| solutions for enrichment bias | --> DILUTE inoculum + grow multiple independent enrichment cultures --> separate organisms from inoculum using flow cytometry --> sort individuals CELLS into wells of 96 well plates so no competition occurs |
| Florescence in-situ hybridization (FISH) | label DNA corresponding to 16S rRNA with florescent molecule, hybridize probe to microbes in environmental sample -can make probe rather specific or general! -can use diff. colored probes at same time! -can base probes on specific functional genes instead of 16S rRNA! |
| How to construct a phylogenetic tree from environmental samples | 1) Isolate DNA from a sample 2) Amplify selected genes 3) Obtain sequences of PCR fragments 4) Identify organisms using sequences, use this to create tree of known/unknown organisms --> usually we find unknown!! |
| single gene analysis vs metagenomics | Single gene phylogenetic tree: comes from community sampling approach, we amplify and sequence a single gene then construct a tree --> gives a snapshot of phylogenetic relationships in community + can identify novel 16S types/species Total gene pool of community: uses an environmental genomics approach, make a genomic library using shotgun sequencing or sequence entire genome using high throughput DNA sequencer, then do assembly of contigs and annotation of partial genomes --> get total gene pool of community with all gene categories, new genes, and links to 16S sequences |
| shortcomes of metagenomics | can be difficult to assign genes to correct organism in community, in a particular environment an organism likely isn't expressing all of its genes |
| Metatranscriptomics & Metaproteomics | "What are they doing?" collect total RNA of proteins (for <20 species), reverse transcribe to DNA and sequence to reveal what genes are being transcribed in environment compare observed metaproteomics to predicted metagenomics (very difficult to assign masses to peptides in genome!!) |
| Chemical Assays of major metabolic reactions | also determines what microbes are doing! may or may not use radioactive isotopes --> use killed control to account for abiotic processes that could produce same stuff --> gives rates of reactions occurring in environmental samples, BUT not what organisms are present, or which ones are doing the reaction you have measured --> false positives can come from accidental extra chemicals they use |
| microelectrodes | measure changes in chemical concentrations over really small distances can be used directly in environment! --> reveal what reactions are happening but not who is doing them |
| stable isotope probing | associates reactions with microbes take environmental sample --> feed C13/N15 substrate --> some cells will metabolize it, others will not --> extract DNA --> separate light from heavy using ultracentrifuge --> remove and analyze using PCR of 16s rRNA/metabolic genes or by sequencing whole genome |
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