1
Q
What are the 2 kinds of analyses of microbial communities?
A
- culture-dependent
- culture-independent
2
Q
What are the kinds of culture-dependent analyses?
A
- enrichment
- isolation
3
Q
How does enrichment work?
A
- collect sample to serve as inoculum culture
- place inoculum into selective media
- DNA is extracted
- sequence 16S r RNA to determine community diversity
4
Q
What is selective media?
A
Media specific for organism of interest
5
Q
What is the step needed to enrich methane-oxidizing organisms?
A
Incubate soil chunk in minimal media with methane
6
Q
What happens when consumption of methane is detected?
A
Detected via GC then sample is transferred to fresh media and process is repeated
7
Q
How does isolation work?
A
- extracted our culture from enriched sample
- culture played on agar plate
8
Q
What happens after growth detected in complete isolation?
A
- streak few times under specific carbon source
- mix molten agar with liquid culture and creamy dilutions
9
Q
What is a modern method for isolation?
A
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
10
Q
What is the process in laser tweezers?
A
- 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
11
Q
What is flow cytometry?
A
- 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
12
Q
are the 2 types of microscopic analysis?
A
- general staining methods
- FISH
13
Q
what are the kinds of general staining methods?
A
- fluorescent staining with dyes that bind nucleic acids
- viability staining
- fluorescent proteins as cell tags and reporter genes
14
Q
fluorescent staining with dyes that bind nucleic acids
A
- DAPI dye - binds DNA (eukaryotic)
- SYBR Green I - bright fluorescence for all micro
15
Q
viability staining
A
- differentiates living from dead cells
- cells live = membrane intact = no dye
- cell dead = membrane not intact = dye
16
Q
fluorescent proteins as cell tags and reporter genes
A
- gene for GFP inserted into genome of bacterium
- if GFP expressed, cell fluorescence green when observed with UV microscopy
17
Q
FISH
A
- 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
18
Q
what are the kinds of genetic analysis?
A
pcr
microarrays
omics
19
Q
PCR
A
amplify DNA that is later used in phylogenetic analysis
20
Q
what is useful to distinguish bacteria at species level?
A
- recA and gyrB
21
Q
what is the genes that encode proteins accumulate?
A
mutations faster than genes for r RNA
22
Q
why is 16S r RNA not helpful to identify species?
A
conserved gene
23
Q
MLST
A
- 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
24
Q
what is MLST have good use in?
A
microbiology and used to differentiate strains of various pathogens
25
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
26
what does the ribotype allow?
rapid identification of different species and different strains of species
27
what is another genome fingerprinting method?
- repetitive extragenic palindromic PCR
- multigene and whole genome analyses
28
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
29
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
30
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
31
phylochips
- microarrays designed for biodiversity studies
- developed for screening of microbial communities
- microarrays designed to detect genes for ammonia oxidation, sulfate respiration, nitrogen fixation
32
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
33
application of OMICS
- genomic
- transcriptomic
- metabolomic
- proteomic
34
genetics
- study heredity
- refers to function and composition of single gene
35
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
36
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
37
modern types of sequencing
illumina sequencing
- sample preparation
- cluster generation
- sequencing
- data analysis
38
compare soil organisms and parasites
soil: larger genomes, complex enviro, diff strategies to survive
parasite: small genomes, partially rely on host where they live
39
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
40
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
Bio 322
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