Unit 9 Flashcards
What are the 2 kinds of analyses of microbial communities?
- culture-dependent
- culture-independent
What are the kinds of culture-dependent analyses?
- enrichment
- isolation
How does enrichment work?
- collect sample to serve as inoculum culture
- place inoculum into selective media
- DNA is extracted
- sequence 16S r RNA to determine community diversity
What is selective media?
Media specific for organism of interest
What is the step needed to enrich methane-oxidizing organisms?
Incubate soil chunk in minimal media with methane
What happens when consumption of methane is detected?
Detected via GC then sample is transferred to fresh media and process is repeated
How does isolation work?
- extracted our culture from enriched sample
- culture played on agar plate
What happens after growth detected in complete isolation?
- streak few times under specific carbon source
- mix molten agar with liquid culture and creamy dilutions
What is a modern method for isolation?
Laser tweezers that includes
- laser beam creates force
- force pushes down microbial cell and holds it in place
- laser beam is moved cell moves with it
What is the process in laser tweezers?
- laser beam drags cells down via specific forces
- cells trapped
- trapped cells flushed from capillary into tube
- tube contains sterile media
- place tube at optimal temp to detect growth
What is flow cytometry?
- Counting and examining microscopic particles by suspending them in stream of fluid and passing them through electronic detector
- cytometers assess size, shape and fluorescent properties of single cells
- cells are examination when passing through detector
- machine sorts cells based on measured criteria
are the 2 types of microscopic analysis?
- general staining methods
- FISH
what are the kinds of general staining methods?
- fluorescent staining with dyes that bind nucleic acids
- viability staining
- fluorescent proteins as cell tags and reporter genes
fluorescent staining with dyes that bind nucleic acids
- DAPI dye - binds DNA (eukaryotic)
- SYBR Green I - bright fluorescence for all micro
viability staining
- differentiates living from dead cells
- cells live = membrane intact = no dye
- cell dead = membrane not intact = dye
fluorescent proteins as cell tags and reporter genes
- gene for GFP inserted into genome of bacterium
- if GFP expressed, cell fluorescence green when observed with UV microscopy
FISH
- not GENETIC
- nucleic acid probe is a DNA or RNA oligonucleotide complementary to sequence in target gene or RNA
- probe and target together = hybridize
- nucleic acid probes made fluorescent by attaching dyes to them
- fluorescent probes used to identify organisms that contain sequence complementary to probe
- phylogenetic FISH stains are fluorescing oligonucleotides complementary to sequence of 16 or 18S r RNA
- phylogenetic stains penetrate cells and hybridizes with r RNA directly in ribosome
what are the kinds of genetic analysis?
pcr
microarrays
omics
PCR
amplify DNA that is later used in phylogenetic analysis
what is useful to distinguish bacteria at species level?
- recA and gyrB
what is the genes that encode proteins accumulate?
mutations faster than genes for r RNA
why is 16S r RNA not helpful to identify species?
conserved gene
MLST
- multi loci sequence type
- relies on different housekeeping genes from related organisms that are examined
- housekeeping genes encode essential functions in cells and located on chromosome not the plasmid
- alleles of each genes are assigned a number
- strain assigned an allelic profile
what is MLST have good use in?
microbiology and used to differentiate strains of various pathogens
genome fingerprinting
- rapid approach for evaluating polymorphisms between strains
- fingerprints are DNA fragments from genomes
- gene is PCR amplified and characterized
- characterize 16S r RNA - ribotyping
- diff genes used to generate fingerprint
- based on localization of 16S r RNA genes on genome fingerprinting
- genomic DNA is extracted and digested by restriction enzymes
- fragments are separated via electrophoresis
- fragments are transferred to membrane
- membrane is incubated and labeled 16S r RNA gene probe
- size and number of bands detected generates a specific pattern
what does the ribotype allow?
rapid identification of different species and different strains of species
what is another genome fingerprinting method?
- repetitive extragenic palindromic PCR
- multigene and whole genome analyses
rep-PCR
- based on highly conserved repetitive DNA elements randomly incorporated in bacterial chromosome
- number and positions of these repetitions caries between strains in species
- primers designed to be complementary to elements
- PCR amplification of genomic fragments between elements
- PCR products can be visualized via agarose gel electrophoresis
- pattern profile of fragments that is characteristic for strain in species
multigene and whole genome analyses
- gives insights into large role of HGT in microbial evolution
- reveals dynamic nature of microbial genomes
- enables comparative analysis of presence or absence of gene, order of genes in genome
- whole genome used for metabolic reconstruction and characterization of genetic pathways
microarray
- lab tool serves to detect expression of many genes at the same time
- DNA microarray are microscope slides that contain thousands of tiny spots in defined position
- each spot has known DNA sequence or gene
- DNA microarray slides called gene chips or DNA chips
- DNA molecules attached to each slide act as probes to detect gene expression
phylochips
- microarrays designed for biodiversity studies
- developed for screening of microbial communities
- microarrays designed to detect genes for ammonia oxidation, sulfate respiration, nitrogen fixation
how to create phylochips?
- fix rRNA gene to chip surface
- phylochip designed for specific group of organisms
- assess the diversity of sulfate-reducing organisms in sulfidic enviro
- obtain 16S r RNA from all known SRB and attach to phylochip
- isolate total community DNA from sediment
- PCR amplification and fluorescence labeling of 16S r RNA genes
- hybridize environmental DNA with probes on phylochip
application of OMICS
- genomic
- transcriptomic
- metabolomic
- proteomic
genetics
- study heredity
- refers to function and composition of single gene
genomics
- study genes and functions
- function and composition of genes and their internal relationships
- goal is to identify combined influence of genes on growth and development of organism
J. Craig Venter
- modern genomics
- computational approach helped in sequencing human genome
cut all genome with restriction enzymes
separate PAA electrophoresis
analyze sequence fragment by fragment
time-consuming
modern types of sequencing
illumina sequencing
- sample preparation
- cluster generation
- sequencing
- data analysis
compare soil organisms and parasites
soil: larger genomes, complex enviro, diff strategies to survive
parasite: small genomes, partially rely on host where they live
what was the goal and what was the organism?
- mycoplasma genitalium to mycoplasma laboratorium
- identify minimal set of genes required to sustain life
- rebuild genes synthetically to create a new organisms
measuring microbial activity in nature
- chemical assays -> measure lactate oxidation to assess activity of SRB
- radioactive 14CO2 -> measure intensity of phototrophy in some area
- microsensors -> microneedle embedded in area
- stable isotopic labeling -> 13C and 34S
