Walker lecture 1

Question 1

how are metazoans ands some unicellular eukaryotes species defined?

Answer 1

by reproductive isolation.

Question 2

For microbes including prokaryotes, species definition?

Answer 2

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

Question 3

what is horizontal gene transfer?

Answer 3

DNA can transfer to different microbes which make species differentiation tough.

Question 4

ford doolittle, what he do

Answer 4

figured out horizontal gene transfer

Question 5

what is a species scape?

Answer 5

shows size of organisms relative to amount of organisms. Problem with the one she showed us is is it lacks microbes.

Question 6

t or f, in 1998 how many predicted microbes where there?

Answer 6

5 ^ 30

Question 7

true or false, most probable number (MPN) is a way to count dead bacteria in a sample.

Answer 7

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)

Question 8

species identification: sample, culture, and make single isolates and identifiction using known techniques

Answer 8

i. e. you culture bacteria, pick a single isolate, and gram stain it, etc.
- con is it must be culturable (very view are)

Question 9

what is shotgun cloning?

Answer 9

1. take a sample
2. isolate DNA
3. PCR with universal primers
4. ligate to a vector or do direct sequencing
5. genebank search

Question 10

what do we mean by universal primers in shotgun cloning

Answer 10

we most often use 16s rRNA

Question 11

what are loop regions and stem regions of 16s rRNA?

Answer 11

• loop regions show divergence between species
• stem regions show conserved regions
• stem are less evolved since they are bind the same molecules throughout life.

Question 12

what are the main reasons 16s rRNA a good universal primer?

Answer 12

• 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

Question 13

when aligning sequences how do we know loop regions vs stem regions?

Answer 13

• 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

Question 14

t or f, by aligning sequences you can build a phylogenetic tree

Answer 14

true

Question 15

who is paul herbert?

Answer 15

he wants to sequence every living thing in the world. He uses two molecular clocks to do this (one is 16s rRNA)

Question 16

We can also identify microbes (other than culture and using sequencing) by using specific molecular probes in situ.. explain this

Answer 16

• use probes that can distinguish very specific characteristics of strains
  i. e. we can use a fluorescent dye probe.
• can detect specific proteins

Question 17

We can also identify microbes (other than culture, using sequencing, and molecular probes) by using a denature gel and then electrophoresis… explain this

Answer 17

1. extract DNA from sample
2. PCR amplify it
3. electrophoresis with denaturing agent (urea)
4. samples separate by size due to ssDNA and dsDNA.
5. G + C determines denaturation

Question 18

what is DGGE and TGGE

Answer 18

DGGE -> denaturing gradient gel electrophoresis

TGGE –> temperature graident gel elctrophoresis

Question 19

What is FAME?

Answer 19

another way to identify bacteria species
stands for fatty acid methyl ester

1. take a cultured or non-cultured group and lyse them via saponification
2. methylate it
3. extract and separate fatty acids based on boiling point
4. compare fatty acid bank with what you get

Question 20

Why is FAME great for pharmaceutical companies?

Answer 20

they have huge libraries to compare too. Also each sample is very cheap. FAME is not used for local microbiologists.

Question 21

FAME and DGGE can both look at nanoparticles..

Answer 21

t or f?

Question 22

what are biology plates?

Answer 22

evaluates respiration of bacteria.

Question 23

what is the process of biology plates

Answer 23

1. place unknown samples in wells full of substrates
2. if respiration is occurring, a dye is released and we see a purple colour
3. cheap and time-efficient … finished in 24 hours

Question 24

What is ElISA?

Answer 24

enzyme-linked immunosorbent assay. Uses an antigen/antibody system. fluorescent dye is coupled with these and indicate a positive rxn

Question 25

what is EISA used for?

Answer 25

food industry to find out what microbes are responsible for spilling food. Similar to biology plates which looks for respiration in spoiled foods.

Question 26

DNA microarrays / gene chips: explain these

Answer 26

1. extract DNA and label it with fluorescence.

2. then hybridize it to a glass slide / array

Question 27

what is a phylochip?

Answer 27

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.

Question 28

what is metagenomics / functional metagenomics

Answer 28

meta –> sequencing all DNA

functional –> sequencing all RNA

Question 29

who was the father of microbial ecology?

Answer 29

van Niel

• he studied electrophoresis that did not result in the production of oxygen
• came up with the phrase everything is everywhere

Question 30

true or false, everything from a living cell can be used as a carbon source

Answer 30

true, van niel figured this shit out