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Michaelmas Term 1 > The Prokaryotes II > Flashcards

The Prokaryotes II Flashcards

1
Q

A species should be

A

Genetically and phenotypically cohesive, and their traits should be distinct from other species

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2
Q

Monophyletic

A

Strains within a species should all share a recent common ancestor, to the exclusion of other species

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3
Q

Phylogenetic tree

A

A diagram or theoretical model of the sequence of evolutionary divergence of species or other groups of organisms from their common ancestors

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4
Q

Give an example of a bacterial species for which HGT is very common and very promiscuous

A

H. pylori

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5
Q

What does it mean for a bacterial species that had very common and promiscuous HGT

A
  • All bacteria share a common gene pool
  • (my thoughts): large pangenome
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6
Q

What happens to a bacterial species that has very uncommon and confined intra-species exchange

A
  • Very little mixing of gene pools
  • taxonomic unit could be individual cell or clone
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7
Q

Give an example of a species for which HGT is very uncommon and confined to intra-species exchange

A

M. leprae

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8
Q

What happens to a bacterial species if HGT is common but confined to intra-species exchange

A

Reproductive isolation and mixing of ‘species’ gene pools would give the equivalent of the Biological Species Concept

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9
Q

How much of the E. Coli genome is derived from lateral gene transfer?

A

10-15%

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10
Q

What are the phonetic (morphological) characters used for classification

A

Only a few:
- Gran stain
- motility

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11
Q

How are phenetic characters gathered?

A
  • non-weighted numerical taxonomy
  • many tests, scored +/-
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12
Q

Describe phenetic characteristics

A

often biochemical (derived from functional genes)

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13
Q

Give examples of phenetic characters

A
  • ability to use single C sources for growth (Biolog, API strips)
  • ability to grow at a range of pH/ temperatures
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14
Q

Which types of tests are suitable to assess phenetic characters?

A

Any test that provides large amounts of data:
- FAME
- gas chromatography
- MALDI-TOF

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15
Q

FAME

A
  • fatty acid methyl ester
  • fatty acid fingerprint
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16
Q

Gas chromatography

A

Detection of metabolites

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17
Q

rRNA is assumed to be

A
  • monophyletic
  • never participated in lateral gene transfer
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18
Q

Why is rRNA assumed to be monophyletic?

A
  • large
  • essential for life; highly conserved
  • difficult for homologous recombination to successfully integrate the sequence
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19
Q

How long is 16s rRNA

A

1500bp

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20
Q

If the assumptions we hold about rRNA are true, then the rRNA sequence is:

A
  1. A universal chronometer suitable for all organisms
  2. A chronometer suitable for very long and very short evolutionary times
  3. A chronometer suitable able for bacterial phylogeny, irrespective of HGT
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21
Q

Changes in rRNA sequence can be used to infer

A

Phylogeny

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22
Q

What is metagenomic gene mining?

A

Pooled nucleic acid is analysed from samples directly from the environment (have not be identified/grown in a lab)

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23
Q

Metagenome

A
  • DNA
  • total gene content of a microbial community
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24
Q

Transcriptome

A
  • RNA
  • total RNA produced by an organism
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25
Q

Proteome

A
  • protein
  • total protein produced by an organism
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26
Q

Microbial species definition

A
  • A group of strains that share certain characteristics traits, are genetically cohesive and share a recent common ancestor
  • The majority of genes in the species have congruent phylogenies and share a recent common ancestor
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27
Q

‘In practice’ definition

A

a combination of:
- DDH cut off at 70%
- a common phenotypic trace

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28
Q

DDH

A

DNA-DNA hybridisation

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29
Q

Strain designation is often related to

A

Pathogenicity

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30
Q

Give an example of two differing bacterial strains

A
  • E. Coli K12 = benign
  • E. Coli O157:H7 = produces enterotoxins
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31
Q

Strain type may relate to a

A

Single gene product

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32
Q

Epidemiology

A
  • The study of the occurrence, distribution and determinants of health and disease in populations
  • how bacterial strains move about
33
Q

PCR

A
  • primers designated to target specific DNA portion
  • dénaturation separates DNA strands
  • primers anneal to complementary sites
  • DNA Polymerase extends DNA sequence following primers
  • 25-35 cycle repeats
34
Q

PCR components:

A
  1. DNA sample
  2. Primers
  3. Nucleotides
  4. Taq polymerase
  5. Mix buffer
  6. PCR tube
35
Q

PCR process

A
  1. Denaturing
  2. Annealing
  3. Extension
36
Q

What does PCR result in?

A

Exponential DNA amplification

37
Q

Quantitative PCR aka

A

Real-time PCR (rtPCR)

38
Q

Quantitative PCR

A
  • monitors amplification of product during amplification cycles
  • SYBR intercalâtes with DNA
  • TaqMan probes contain fluorescent reporters
  • more accurate than end-point analysis w gel electrophoresis
39
Q

FISH acronym

A

Fluorescence In Situ Hybridisation

40
Q

FISH

A
  • specific probes designed and tagged with fluorophores
  • target DNA attached to substrate
  • probe and target DNA incubated for ~12 hours for hybridisation
  • excess probe washed away
  • results viewed under fluorescence microscope
41
Q

Give an example of a substrate used in FISH

A

Glass

42
Q

What is FISH used for?

A
  • bacterial speciation using 16S rRNA target
  • genetic counselling
  • specific RNA in tumour cells, tissue etc
43
Q

What is genetic counselling?

A

Identification of genetic disorders

44
Q

What is Sanger sequencing based on?

A

random incorporation of chain-terminating dideoxynucleotides

45
Q

How does Sanger sequencing work?

A

The different lengths of nucleotide are separated, and the labelled nucleotide detected gives the DNA sequence

46
Q

What equipment does Sanger sequencing use?

A
  • ABI 3700 automated DNA analyser
  • capillary array
  • computer (for output)
47
Q

Give two examples of next generation sequencing

A
  1. Illumina MiSeq system
  2. Flow Cell
48
Q

What does next generation sequencing do?

A

Tracks the addition of labelled nucleotides as the DNA chain is copied

49
Q

How is next generation sequencing conducted, and what does it give?

A
  • parallel sequencing
  • massive data output
50
Q

MLST basics

A
  • Multilocus Sequence Typing
  • gene-by-gene approach
51
Q

MLST specifics

A
  • portions of 7 housekeeping genes are sequenced from the bacterial genome
  • each different sequence is assigned an allele number
  • combination of 7 allele numbers is given a sequence type
52
Q

How big are portions sequenced in MLST?

A

~500bp

53
Q

From which part of the genome are the housekeeping genes taken and why?

A
  • Core genome
  • stable
54
Q

Give the MLST core genome sequence type for Campylobacter

A

1343

55
Q

Combination of 7 allele numbers is known as

A

Allelic profile

56
Q

PubMLST

A

stores info for both DNA sequence and isolates

57
Q

Compare and contrast cloning and PCR

A

Cloning: within cells
PCR: in vitro

58
Q

Describe molecular cloning

A
  1. Choose prokaryote or eukaryote cloning and vector system
  2. Restriction ending lease cuts DNA
  3. DNA Ligase joins new piece of foreign DNA to plasmid vector
  4. Cells made competent to receive DNA using heat shock/electroporation
59
Q

What are the two options for a prokaryotic cloning and vector system?

A
  1. E. Coli
  2. Bacillus subtilis
60
Q

What is the eukaryotic cloning and vector system?

A

Saccharomyces cerevisiae

61
Q

What is it important to do after molecular cloning?

A

Screen for transformed cells

62
Q

Applications of molecular cloning

A
  • look for expression, organisation, function and importance of a particular gene
  • produce recombinant proteins or subunit vaccines
  • transgenic organisms
63
Q

Give an example of a recombinant protein created using cloning

A

Insulin

64
Q

Give an example of a subunit vaccine produced using cloning

A

Hepatitis B vaccine

65
Q

What is the advantage of a subunit vaccine?

A

Patients can be immunised without direct exposure to pathogen

66
Q

Give examples of useful transgenic organisms made using cloning

A
  • knockout mice for research
  • crops (wheat, soybean, cotton, canola) with herbicide, insectide or antimicrobial resistance
67
Q

When was the first isolation of insulin?

A
  • Banting/Best and Macleod/Collip
  • Nobel Prize 1923
68
Q

When did Frederick Sanger win the Nobel Prize?

A

1958

69
Q

Describe human insulin protein

A
  • 51 aas
  • 2 peptide chains cross linked by disulphide bridges
70
Q

When was the first synthetic insulin commercially available?

A

1982

71
Q

What is the function of insulin

A
  • produced by pancreases in response to high blood sugar level
  • promotes glucose absorption from blood into liver, fat and muscle cells
72
Q

Type I Diabetes mellitus

A

Insulin producing cells destroyed by autoimmune reaction

73
Q

Type II Diabetes mellitus

A
  • Insufficient insulin produced by pancreas
  • predisposing factors: diet, genetics
74
Q

How is human insulin synthetically produced?

A
  • human insulin producing gene isolated from human pancreas felll, and inserted into plasmid DNA from bacterial cell cut with restriction enzymes to create recombinant DNA
  • recombinant DNA introduced into bacterial cell, and put in fermentation tank to multiply
  • human insulin extracted and purified
75
Q

Future insulin needs

A

from 11 billion dollars in 2014 to 54bn in 2024

76
Q

What does highly conserved mean?

A

very little change, very slowly over time

77
Q

Raw genome sequencing

A

Sequence the whole genome

78
Q

‘Contained use’

A

Control measures such as physical, chemical, or biological barriers used to limit contacts between GMOs and humans / the environment

Michaelmas Term 1 (42 decks)

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