Walker lecture 1 Flashcards
how are metazoans ands some unicellular eukaryotes species defined?
by reproductive isolation.
For microbes including prokaryotes, species definition?
often defined by sequence identity often using 16s RNA. clinicians don’t live by this since often two strains with 99% similarity will have different envelope proteins which causes different disease
what is horizontal gene transfer?
DNA can transfer to different microbes which make species differentiation tough.
ford doolittle, what he do
figured out horizontal gene transfer
what is a species scape?
shows size of organisms relative to amount of organisms. Problem with the one she showed us is is it lacks microbes.
t or f, in 1998 how many predicted microbes where there?
5 ^ 30
true or false, most probable number (MPN) is a way to count dead bacteria in a sample.
false, it counts live bacteria similarly to CFU.
to count dead we use optical density.
real time PCR is a way to measure dead, living, respiratory microbe (anything)
species identification: sample, culture, and make single isolates and identifiction using known techniques
i. e. you culture bacteria, pick a single isolate, and gram stain it, etc.
- con is it must be culturable (very view are)
what is shotgun cloning?
- take a sample
- isolate DNA
- PCR with universal primers
- ligate to a vector or do direct sequencing
- genebank search
what do we mean by universal primers in shotgun cloning
we most often use 16s rRNA
what are loop regions and stem regions of 16s rRNA?
- loop regions show divergence between species
- stem regions show conserved regions
- stem are less evolved since they are bind the same molecules throughout life.
what are the main reasons 16s rRNA a good universal primer?
- present in all microbes (16s for microbes, 18s for eukaryotes)
- 2000 BP long
- both slowly and rapidly evolving (stem vs. loop)
- horizontal gene transfer of 16sRNA is rare
- large data base of them
- they can be aligned due to their conservation
when aligning sequences how do we know loop regions vs stem regions?
- sequences with similarities are stem
- sequences with differences are loops
this alignment can only be truly done once you know the general structure of the RNA theyre derived from
AAAUCCGU
AUCUCCGG
SLL S SSSL
t or f, by aligning sequences you can build a phylogenetic tree
true
who is paul herbert?
he wants to sequence every living thing in the world. He uses two molecular clocks to do this (one is 16s rRNA)
We can also identify microbes (other than culture and using sequencing) by using specific molecular probes in situ.. explain this
- use probes that can distinguish very specific characteristics of strains
i. e. we can use a fluorescent dye probe. - can detect specific proteins
We can also identify microbes (other than culture, using sequencing, and molecular probes) by using a denature gel and then electrophoresis… explain this
- extract DNA from sample
- PCR amplify it
- electrophoresis with denaturing agent (urea)
- samples separate by size due to ssDNA and dsDNA.
- G + C determines denaturation
what is DGGE and TGGE
DGGE -> denaturing gradient gel electrophoresis
TGGE –> temperature graident gel elctrophoresis
What is FAME?
another way to identify bacteria species
stands for fatty acid methyl ester
- take a cultured or non-cultured group and lyse them via saponification
- methylate it
- extract and separate fatty acids based on boiling point
- compare fatty acid bank with what you get
Why is FAME great for pharmaceutical companies?
they have huge libraries to compare too. Also each sample is very cheap. FAME is not used for local microbiologists.
FAME and DGGE can both look at nanoparticles..
t or f?
what are biology plates?
evaluates respiration of bacteria.
what is the process of biology plates
- place unknown samples in wells full of substrates
- if respiration is occurring, a dye is released and we see a purple colour
- cheap and time-efficient … finished in 24 hours
What is ElISA?
enzyme-linked immunosorbent assay. Uses an antigen/antibody system. fluorescent dye is coupled with these and indicate a positive rxn
what is EISA used for?
food industry to find out what microbes are responsible for spilling food. Similar to biology plates which looks for respiration in spoiled foods.
DNA microarrays / gene chips: explain these
- extract DNA and label it with fluorescence.
2. then hybridize it to a glass slide / array
what is a phylochip?
a specific array which is used to identify microbes from environmental samples (in DNA micro-arraying). it is essentially a chip with many sequences which hybridize samples.
what is metagenomics / functional metagenomics
meta –> sequencing all DNA
functional –> sequencing all RNA
who was the father of microbial ecology?
van Niel
- he studied electrophoresis that did not result in the production of oxygen
- came up with the phrase everything is everywhere
true or false, everything from a living cell can be used as a carbon source
true, van niel figured this shit out
