Lecture 1 Flashcards
What does the science of microbial ecology focus on?
Focuses on how microbial populations assemble to form communities and how these communities interact with each other and with their environment
What are examples of culture independent analyses of microbial communities?
-General staining methods
-Fluorescent In Situ Hybridization (FISH)
-PCR Methods of Microbial Community Analysis
-Environmental Genomics and Related Methods
What is DAPI? What is it used for?
4’, 6-diamidino-2-phenylindole is a general fluorescent stain that binds strongly to adenine-thymine (AT) rich regions in DNA nad thus stains DNA.
It is used for identifying microorganisms in natural samples
-Live cells ~ green ( cells w an intact membrane are alive)
-Dead cells ~ red (cells with a compromised membrane are dead)
How does live bacterial gram stain kit work?
The dye binds specifically to N-acetylglucosamine in the peptidoglycan layer of gram pos bacteria.
What does the live bacterial gram stain kit contain?
~Contains WGA (Wheat Germ Agglutinin) conjugated to CF594 to stain the surface of gram pos cells with RED fluorescence.
~The kit also includes DAPI to stain the DNA of all bacteria with blue fluorescence.
What is WGA?
Fluorescently-labeled wheat germ agglutinin.
WBA is a carbohydrate-binding lectin that has a high affinity for sialic acid and N-acetylglucosamine
What way will gram pos and gram neg bacteria be stained using this live bacterial gram stain kit?
-Gram pos bacteria will show a fluorescent blue interior and fluorescent red cell
-Gram neg bacteria will be stained w blue fluorescence only
What is the purpose of viability PCR dyes?
PMAxx
-Membrane impermeable so they are dead cell specific
-Once inside of a dead cell, they bind to DNA
-Exposure to intense visible light renders the dyes reactive and causes them to covalently attach to the DNA
-This DNA modification prevents amplication is subsequent PCR reactions
What unique feature makes PMAxx highly useful in selective detection of live bacteria by PCR?
In a population of live and dead cells, only dead cells are susceptible to DNA modification due to compromised cell membranes.
What are the 5 steps in v-PCR?
-Dye addition
-Incubation
-Light exposure
-DNA extraction
-qPCR (quantitative PCR)
(No PCR amplification in DEAD cells)
What is the difference between bioluminescent and fluorescent bioluminescent?
Bioluminescent - glows in the dark
Fluorescent bioluminescent - glows in response to UV light
-e.g Aequorea victoria is a crystal jellyfish that is both
Why does the jellyfish Aequorea victoria fluoresce?
GFP (green fluorescent protein) is a naturally occuring 27kDa protein derived from the jellyfish Aequorea victoria that fluoresces green when exposed to blue light
The GFP gene can be cloned on a vector and transformed into diff bacteria e.g Bacillus megaterium - the GFR makes cells autofluorescent and is a means of tracking cells introduced into the environment.
What is C.elegans?
C.elegans is a free-living nematode transparent nematode about 1mm in length that lives in temperate soil environments
Main principles of PCR?
-Forgoes the need for culture of microbes
-No isolations of microbes
-No microscopic identification or quantification
What can PCR be used to identify?
-Specific genes are used as measures of biodiversity and metabolic capacity
-Some genes are unique to an organism so detection in sample implies that organism is represented in sample
-Examination of gene expression in sample implies those organisms are metabolically acrive and pinpoints a pathway that is active under those particular conditions
Pro of PCR methods of microbial community analysis?
-Many phylogenetically distinct microbes are present in a population - this allows phylotypes (organisms that are closely related) to be described
How much of bacteria have been cultured?
Less than 0.1%
What is PCR called a chain reaction?
Polymerase Chain Reaction
-because the rxn continuously repeats until the primers are used up - yield millions of copies of a simple single fragment for each primer pair
Process of PCR
1) Denaturation - the rxn is heated to 94-98 degrees C for 20-30 seconds to break the hydrogen bonds between the strands
2) Annealing - the reaction temp is lowered to 50-60 degrees C for 20-40 seconds to allow primers to anneal to the template strands
3) Elongation - the temp is increased (optimal temp dependent on DNA Polymerase used e.g 72-78 C for Taq Polymerase) to allow for the addition of dNTPs.
The amopunt of target sequence doubles with each thermal cycle which leads to an exponential amplification represented by 2.
What is phylogenetics?
The analysis of molecular sequencing data to study evolutionary relationships among groups of organisms
What is phylogeny?
The evolutionary history of a group of organisms
What is evolution?
A process of inherited nucleotide sequence change, comparative analyses of DNA sequences allow the reconstruction of phylogenetic histories
What are rRNA genes and why are they targeted in phylogenetic analysis?
-rRNA genes are the RNA component of the ribsome which is involved in protein synthesis
-Used as they are universally distributed, functionally constant and sufficiently conserved (slow changing)
Who proposed the use of small subunits of rRNA genes as targets for phylogenetic analysis?
Carl Woese
What rRNA species are used in pro and euk for phylogenetic analysis?
Pro - 16S rRNA is the right size - from small 30S ribosomal subunit
Euk - 18S rRNA
Loops vs Stems
Loops - sites that are more free to mutate and evolve faster
Stems - sites that rarely mutate and are conserved (stay same)
Components of 16S rRNA?
Consists of 9 variable regions and is approx 1500 base pairs long
-conserved regions - unspecific applications
-variable regions - group or species-specific applications
