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mcrb 265 > microbial genomics > Flashcards

microbial genomics Flashcards

1
Q

what is human genome projecT?

A

1990 - goal to sequence al 3*10^9 base pairs of human DNA

- completed in 2003 - thought it would take 30 year.s

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2
Q

who did HGP?

A

public: US govt, Francis Collin
private: The institute Craig venter

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3
Q

first completed genome sequence was?

A

haemophilus influenzae

- respiratory disease. took 8 years thru private funding

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4
Q

venter style?

A
  • break dna into pieces, sequence all at same time. computer sees where overlap + sticks together where overlaps.
    = contigs : overlap
    = less accurate bc gaps btw regions + repetitive sequences.
    but cheap + fast
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5
Q

collins style?

A

simplistic approach
- sequence DNA. find end of gene. create probe that extended from end of piece to start of next piece

slow, systematic. had to create probe between each finding.

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6
Q

sequencing DNA

A

sequence thousands of clones at one. 7-10 times coverage of entire genome

  • assembles contigs using computer algorithms
  • fill in gaps using targeted methods - chromosomal walking
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7
Q

what’s sanger sequencing?

A

DNA polymerase in soln copies after primer.

di-deoxynucleotide (no -OH) addd to soln. DNApol can’t copy if no -OH.
separate based on size. know based on dideoxy what original nucleotide is.
shorter strands at beginning of sequence

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8
Q

next gen sequencing

A
  • increase in amount of genome/metagenome sequence. (many sequences at a time)
  • non-specialist labs able to use (signal when nucleotides incorporated; quick, cheap)
  • useful for resequencing (comparative - looking for disease)
  • shotgun sequencing + computer power.
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9
Q

structure of ORF

A

open reading frame
- approx 300 bp before stop codon.
ORF =/= gene. may be, but doesnt need to be. if transcribed + translated = gene.

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10
Q

using ORF

A
  1. comp finds codons
  2. computer finds possible stop codons
  3. combuter counts codons between start + stop.
  4. computer finds possible RBS
  5. computer calculares codon bias in ORF
  6. computer decides if likely to be genuine
  7. comp gives list of probably ORF
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11
Q

ORF content to genome size

A

greater genome size = more ORF content

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12
Q

lifestyles of bacteria

A

endosymbiotic - live in other cell. use host DNA, but have their own

parasitic: may grow inside cell. can’t grow without host DNA

Free-living: independent. dont need other organisms. usually larger than others. fitness cost

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13
Q

gene annotation

A

compare ORF, if similar sequence annotated as similar function.
- gene annotations help reconstruct metabolic pathways + determine gene complement

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14
Q

problem with gene annotation?

A

2 sequences may have diff function

- if dont have sequence similar, maybe protein is still same function

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15
Q

pathogens + growth factors

A

from host cell.
no genes for amino acid biosynthesis
- no genes

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16
Q

what is URF

A

ORF with unknown function

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17
Q

Re-constructed Genome map

A

ORF’s in opposite directions.

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18
Q

analyze map of bacteria + determine what kind of bacteria 1

A
  • sugar transport
  • peptide transport
  • flagellum
  • NH4+
    Zinc, Fe.

glycolysis, PPP
fermenter. lactate, no O2

19
Q

analyze map of bacteria + determine what kind of bacteria 2

A 
TCA cycle. 
has ETC
flagella, 
pilli
have NADH oxidizer
20
Q

analyze map of bacteria + determine what kind of bacteria 3

A

calvin cycle, carbon fixation.
citric acid cycle
photosynthetic -
NH3 + O2 at 1st etc,

ammonia-oxidizing bacterium.
chemolithoautotroph
aerobe

21
Q

ORFs in bacterial genome : genome vs function

A

DNA replication: same amount needed regardless of genome. proportionally decrease tho. same with translation

trancription + signal transduction : more regulation, more pathways when there’s bigger size.

energy: about stable - increase genome doesnt change energy much

22
Q

what are homologous genes

A

from same ancestor

23
Q

diff btw paralog and ortholog

A

paralog: gene duplication: gene stays within same species or organism (a and b-heme)
- 1 gene retains function, other may vary/pseudogene.

  1. ortholog: copies of same gene, split into diff species same function in 2 diff species.
24
Q

define synteny

A

similar gene order

- more closely related = more syntenous

25
Q

core genome vs acnillary genome vs pan genome

A

core: shared by all strains of same species (specific to group of interest)
ancillary: foudn in some strains of same species (one’s not common to all)
pan: all genes found in group

26
Q

looking for HGT - look for?

A

gene islands: GC diff than rest of chromosome, codon usage bias. flanked by inverted repeats. transferred by HGT
transposons: move around, inverted repeats.
pathogenicity islands: turn normal into virulent factor block chromosomal genes that encode virulense

27
Q

what is metagenoics

A

focus on individual genes previously.

-all genes in enviro. extract DNA and reconstruct community

28
Q

ocean metagenomic analysis

A

look for DNA from cloned genes linked to rRNA gene to ID what organisms might be doing

SR: abundant, couldnt culture.. found RNA gene in it + sequenced - related to rhosopsin.
- uses light energy to pump Cl- out.

when put that gene into e.coli - could pump H+ across using light. photoheterotrophs use to gain more energy from food they have. little nutrient in enviro, light gives energy

29
Q

human microbiome size

A

10-100 trillion bacteria mostly in gut.

connection btw human health + illness

30
Q

bacteroides and obesity

A

bacteroides + firmicutes = break down food.
obese: less bacteroides + more firmicutes than slim ppl
bacteroides increase as obese ppl lose weight

31
Q

diff btw bacteroides + firmicutes?

A

firmicutes get more energy out of food =

32
Q

sequence analysis: bioinformatics

A 
computational analysis of genome data 
- genome content, structure, arrangement
- predict protein structure + function
- produce annotation to determine that location of genes (databases contain gene info)
 = in silico analysis.
33
Q

reverse transcriptase

A

enzyme from retrovirus used to produce DNA copies “cDNA” of their genome to integrate into host genome

34
Q

comparative genomics

A

look at groups of organisms relate to each other.

- ID the overlaps in genome. find core + ancillary genomes

35
Q

mutations in E.coli - evolutionary pressures

A

150000 generations + Id’d similarities.ID’d mutations in relation to ancestral condition

36
Q

Total mRNA

A

extracted from control + experimental cells to determine changes in gene expression

37
Q

creation of genomic microarray + creation of labeled probe representing all mRNA in cell at time

A

genomic DNA -> PCR -> probe hybridization -> signal detection -> data analysis

probe hybridization: make cDNA porbe and label

38
Q

Microarraws: detecting differential gene expression

A

differently labeled cDNA from each culture mixed + hybridized to array.
- labeled probe finds complementary sequence. hybridizes by normal DNA base-pairing rules
- fluorescent signal detected where probe is hybridized.
= require several replications and verigication by other methods

39
Q

Transcriptome of P.aeruginosa

A
  • assemblage of microarray data

- global gene expression from cells grown wiwith hgih vs low levels of Ca2+

40
Q

RNa sequence

A

next gen - avoid microarray

  • sequences transciptome with rev.transcripted cDNA
  • quantitatively determine copy numbers of genes - tell what’s up/down regulated in experimental relative to control
41
Q

what are proteomics?

A

dtermining differences in translation

= what proteins are being made?

42
Q

proteome?

A

proteins present in cell, tissue, or organism at any one point in time

43
Q

mRNA copies in glycolysis vs sporulation

A

glycolysis, some genes on, some off.
sporulation - all increase
diff regulation to diff levels

44
Q

proteomics interactome

single cell genomics

A
  • > all proteins being produced in community.

- > looking at genome only within one cell

mcrb 265 (14 decks)

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