Karteikarten
Lecture 25 PMB
Karten in diesem Stapel (14)
- 1Winogradsky columnartificial 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!! - 2Developing enrichment culturesuse 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)
- 3Results of enrichment culturesPositive 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 - 4enrichment biasin liquid enrichment cultures, most dominant organisms are the ones that grow most rapidly EVEN IF they are not most abundant in actual environment
- 5reasons for enrichment bias1) 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 - 6solutions 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 - 7Florescence 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! - 8How to construct a phylogenetic tree from environmental samples1) 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!! - 9single gene analysis vs metagenomicsSingle 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 - 10shortcomes of metagenomicscan be difficult to assign genes to correct organism in community,
in a particular environment an organism likely isn't expressing all of its genes - 11Metatranscriptomics & 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!!) - 12Chemical Assays of major metabolic reactionsalso 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 - 13microelectrodesmeasure 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 - 14stable isotope probingassociates 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