The Prokaryotes II Flashcards
A species should be
Genetically and phenotypically cohesive, and their traits should be distinct from other species
Monophyletic
Strains within a species should all share a recent common ancestor, to the exclusion of other species
Phylogenetic tree
A diagram or theoretical model of the sequence of evolutionary divergence of species or other groups of organisms from their common ancestors
Give an example of a bacterial species for which HGT is very common and very promiscuous
H. pylori
What does it mean for a bacterial species that had very common and promiscuous HGT
- All bacteria share a common gene pool
- (my thoughts): large pangenome
What happens to a bacterial species that has very uncommon and confined intra-species exchange
- Very little mixing of gene pools
- taxonomic unit could be individual cell or clone
Give an example of a species for which HGT is very uncommon and confined to intra-species exchange
M. leprae
What happens to a bacterial species if HGT is common but confined to intra-species exchange
Reproductive isolation and mixing of ‘species’ gene pools would give the equivalent of the Biological Species Concept
How much of the E. Coli genome is derived from lateral gene transfer?
10-15%
What are the phonetic (morphological) characters used for classification
Only a few:
- Gran stain
- motility
How are phenetic characters gathered?
- non-weighted numerical taxonomy
- many tests, scored +/-
Describe phenetic characteristics
often biochemical (derived from functional genes)
Give examples of phenetic characters
- ability to use single C sources for growth (Biolog, API strips)
- ability to grow at a range of pH/ temperatures
Which types of tests are suitable to assess phenetic characters?
Any test that provides large amounts of data:
- FAME
- gas chromatography
- MALDI-TOF
FAME
- fatty acid methyl ester
- fatty acid fingerprint
Gas chromatography
Detection of metabolites
rRNA is assumed to be
- monophyletic
- never participated in lateral gene transfer
Why is rRNA assumed to be monophyletic?
- large
- essential for life; highly conserved
- difficult for homologous recombination to successfully integrate the sequence
How long is 16s rRNA
1500bp
If the assumptions we hold about rRNA are true, then the rRNA sequence is:
- A universal chronometer suitable for all organisms
- A chronometer suitable for very long and very short evolutionary times
- A chronometer suitable able for bacterial phylogeny, irrespective of HGT
Changes in rRNA sequence can be used to infer
Phylogeny
What is metagenomic gene mining?
Pooled nucleic acid is analysed from samples directly from the environment (have not be identified/grown in a lab)
Metagenome
- DNA
- total gene content of a microbial community
Transcriptome
- RNA
- total RNA produced by an organism
Proteome
- protein
- total protein produced by an organism
Microbial species definition
- 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
‘In practice’ definition
a combination of:
- DDH cut off at 70%
- a common phenotypic trace
DDH
DNA-DNA hybridisation
Strain designation is often related to
Pathogenicity
Give an example of two differing bacterial strains
- E. Coli K12 = benign
- E. Coli O157:H7 = produces enterotoxins
Strain type may relate to a
Single gene product
Epidemiology
- The study of the occurrence, distribution and determinants of health and disease in populations
- how bacterial strains move about
PCR
- 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
PCR components:
- DNA sample
- Primers
- Nucleotides
- Taq polymerase
- Mix buffer
- PCR tube
PCR process
- Denaturing
- Annealing
- Extension
What does PCR result in?
Exponential DNA amplification
Quantitative PCR aka
Real-time PCR (rtPCR)
Quantitative PCR
- 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
FISH acronym
Fluorescence In Situ Hybridisation
FISH
- 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
Give an example of a substrate used in FISH
Glass
What is FISH used for?
- bacterial speciation using 16S rRNA target
- genetic counselling
- specific RNA in tumour cells, tissue etc
What is genetic counselling?
Identification of genetic disorders
What is Sanger sequencing based on?
random incorporation of chain-terminating dideoxynucleotides
How does Sanger sequencing work?
The different lengths of nucleotide are separated, and the labelled nucleotide detected gives the DNA sequence
What equipment does Sanger sequencing use?
- ABI 3700 automated DNA analyser
- capillary array
- computer (for output)
Give two examples of next generation sequencing
- Illumina MiSeq system
- Flow Cell
What does next generation sequencing do?
Tracks the addition of labelled nucleotides as the DNA chain is copied
How is next generation sequencing conducted, and what does it give?
- parallel sequencing
- massive data output
MLST basics
- Multilocus Sequence Typing
- gene-by-gene approach
MLST specifics
- 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
How big are portions sequenced in MLST?
~500bp
From which part of the genome are the housekeeping genes taken and why?
- Core genome
- stable
Give the MLST core genome sequence type for Campylobacter
1343
Combination of 7 allele numbers is known as
Allelic profile
PubMLST
stores info for both DNA sequence and isolates
Compare and contrast cloning and PCR
Cloning: within cells
PCR: in vitro
Describe molecular cloning
- Choose prokaryote or eukaryote cloning and vector system
- Restriction ending lease cuts DNA
- DNA Ligase joins new piece of foreign DNA to plasmid vector
- Cells made competent to receive DNA using heat shock/electroporation
What are the two options for a prokaryotic cloning and vector system?
- E. Coli
- Bacillus subtilis
What is the eukaryotic cloning and vector system?
Saccharomyces cerevisiae
What is it important to do after molecular cloning?
Screen for transformed cells
Applications of molecular cloning
- look for expression, organisation, function and importance of a particular gene
- produce recombinant proteins or subunit vaccines
- transgenic organisms
Give an example of a recombinant protein created using cloning
Insulin
Give an example of a subunit vaccine produced using cloning
Hepatitis B vaccine
What is the advantage of a subunit vaccine?
Patients can be immunised without direct exposure to pathogen
Give examples of useful transgenic organisms made using cloning
- knockout mice for research
- crops (wheat, soybean, cotton, canola) with herbicide, insectide or antimicrobial resistance
When was the first isolation of insulin?
- Banting/Best and Macleod/Collip
- Nobel Prize 1923
When did Frederick Sanger win the Nobel Prize?
1958
Describe human insulin protein
- 51 aas
- 2 peptide chains cross linked by disulphide bridges
When was the first synthetic insulin commercially available?
1982
What is the function of insulin
- produced by pancreases in response to high blood sugar level
- promotes glucose absorption from blood into liver, fat and muscle cells
Type I Diabetes mellitus
Insulin producing cells destroyed by autoimmune reaction
Type II Diabetes mellitus
- Insufficient insulin produced by pancreas
- predisposing factors: diet, genetics
How is human insulin synthetically produced?
- 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
Future insulin needs
from 11 billion dollars in 2014 to 54bn in 2024
What does highly conserved mean?
very little change, very slowly over time
Raw genome sequencing
Sequence the whole genome
‘Contained use’
Control measures such as physical, chemical, or biological barriers used to limit contacts between GMOs and humans / the environment
