Lecture 25 PMB

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Ecology
Candice Young
Flashcards by Candice Young, updated more than 1 year ago
Candice Young
Created by Candice Young over 6 years ago
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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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