Experimental Molecular Evolution
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Transcript of Experimental Molecular Evolution
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Experimental Molecular EvolutionExperimental Molecular Evolution
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Evolution of bacterial resistance to antibiotics
D. M. Weinreich, N. F. Delaney, M. A. DePristo & D. L. Hartl. 2006. Darwinian Evolution Can Follow Only Very Few Mutational Paths to Fitter Proteins. Science 312: 111-114.
Evolution of bacterial resistance to antibiotics
D. M. Weinreich, N. F. Delaney, M. A. DePristo & D. L. Hartl. 2006. Darwinian Evolution Can Follow Only Very Few Mutational Paths to Fitter Proteins. Science 312: 111-114.
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Resistance to ß-lactam antibiotics (e.g., penicillin) is mediated by ß-lactamase, which hydrolyses and inactivates these drugs.
5 point mutations in ß-lactamase jointly increase resistance to ß-lactam antibiotics by a factor of ~100,000. These consist of four missense mutations (A42G, E104K, M182T, G238S) and one 5' noncoding mutation (g4205a).
5 mutations must occur for the resistant allele TEM* to evolve from the wild type allele TEMwt.
Resistance to ß-lactam antibiotics (e.g., penicillin) is mediated by ß-lactamase, which hydrolyses and inactivates these drugs.
5 point mutations in ß-lactamase jointly increase resistance to ß-lactam antibiotics by a factor of ~100,000. These consist of four missense mutations (A42G, E104K, M182T, G238S) and one 5' noncoding mutation (g4205a).
5 mutations must occur for the resistant allele TEM* to evolve from the wild type allele TEMwt.
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There are 5! = 120 mutational trajectories to evolve TEM* from TEMwt.
Experimental Results:
102 of the 120 mutational trajectories from TEMwt to TEM* are selectively inaccessible.
Most resistance evolved through 10 mutational trajectories.
There are 5! = 120 mutational trajectories to evolve TEM* from TEMwt.
Experimental Results:
102 of the 120 mutational trajectories from TEMwt to TEM* are selectively inaccessible.
Most resistance evolved through 10 mutational trajectories.
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Tree of Life
Hypothesis: Last Universal Common Ancestor (LUCA) was hyperthermophilic (>80 °C), lived in hydrothermal vents (black smokers)
Mesophile = 20-40°C Thermophile = 45-75°C Hyperthermophile ≥ 80°C
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xx x x x xxx x x x xx
>3000 B.C.
Proto-Germanic
MiddleEnglish
Old English
Old HighGerman
Gothicsnaiws
snow
snaw
sneoChurchSlavonic
snegu
Old Irish
Proto-Indoeuropean
Old Norse
Greekφιν
.Old Fr
Latin
OldPrussian
snechte
œsn rnoif
, nix nivus
α
*snigw -h
xx
Slavic Germanic Romance Celtic
Reconstructing the past from the present
Reconstruction says something about the Proto-Indoeuropeans
They lived where it snowed.
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Elongation Factor-Tu: G-protein involved in translationElongation Factor-Tu: G-protein involved in translation
Used to elucidate ancient evolutionary relationships
EF-Tu is thermostable in thermophilic organisms, not in mesophilic organisms
EF-Tu from thermophiles is not optimally functional at mesophilic temperatures
Linear relationship between optimal binding temperature of EF protein and optimal growth temperature of the host organism.
Used to elucidate ancient evolutionary relationships
EF-Tu is thermostable in thermophilic organisms, not in mesophilic organisms
EF-Tu from thermophiles is not optimally functional at mesophilic temperatures
Linear relationship between optimal binding temperature of EF protein and optimal growth temperature of the host organism.
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Proteobacteria
Cyanobacteria
Spirochaete
Green Sulfur
Bacillus
ActinobacteriaThermus
Thermotogale
Outgroup Outgroup
Thermotogale
Thermus
Bacillus
Green Sulfur
Spirochaete
Cyanobacteria
Proteobacteria
Actinobacteria
Maximum Likelihood Tree Alternative Tree
ML-Stem
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Proteobacteria
Cyanobacteria
Spirochaete
Green Sulfur
Bacillus
ActinobacteriaThermus
Thermotogale
Outgroup Outgroup
Thermotogale
Thermus
Bacillus
Green Sulfur
Spirochaete
Cyanobacteria
Proteobacteria
Actinobacteria
Maximum Likelihood Tree Alternative Tree
ML-Stem Alt-Stem
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Proteobacteria
Cyanobacteria
Spirochaete
Green Sulfur
Bacillus
ActinobacteriaThermus
Thermotogale
Outgroup Outgroup
Thermotogale
Thermus
Bacillus
Green Sulfur
Spirochaete
Cyanobacteria
Proteobacteria
Actinobacteria
Maximum Likelihood Tree Alternative Tree
ML-Stem
ML-Meso
Alt-Stem
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Synthesizing Ancestral ProteinsSynthesizing Ancestral Proteins
Generate overlapping primer pairs, extended using PCR (Each primer = 50 bases, with 20 base overlap)
Generate overlapping primer pairs, extended using PCR (Each primer = 50 bases, with 20 base overlap)
Gene inserted into cloning vector and sequenced
Removed from cloning vector, inserted into expression vector and sequenced again
Transformed into expression host (E. coli, ER2566), induced with IPTG
This results in the translation of a fusion construct containing:
- Chitin Binding Domain- Intein- EFTu gene
Gene inserted into cloning vector and sequenced
Removed from cloning vector, inserted into expression vector and sequenced again
Transformed into expression host (E. coli, ER2566), induced with IPTG
This results in the translation of a fusion construct containing:
- Chitin Binding Domain- Intein- EFTu gene
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Precursor
CBD-InteinEF-Tu
111 kDa
66 kDa45 kDa
EF-Tu Antibody
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20 30 40 50 60 70 80 90
oC
Relative amount of [
3H] GDP Incorporation
E. coli
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Relative amount of [
3H] GDP Incorporation
ML-meso
E. coli
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Thermus
oC
Relative amount [
3H] GDP Incorporation
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Thermus
ML-stem
oC
Relative amount [
3H] GDP Incorporation
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Thermus
Alt-stem
ML-stem
oC
Relative amount [
3H] GDP Incorporation
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ThermusAlt-stemML-stemThermotoga
oC
Relative amount [
3H] GDP Incorporation
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HydrothermalVents:
Broad Range of Temperatures
Across Narrow Area
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ThermalHot Springs:
Narrow Range of Temperatures
Across Broad Area
Consistent withancient EFs
~65ºC
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Molecular Molecular BreedingBreeding
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Very variableVery variablepopulationpopulation
MonomorphicMonomorphicpopulationpopulation
selectionselection
++breedingbreeding
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Less variableLess variablepopulationpopulation
NoNopopulationpopulation
selectionselection
++breedingbreeding
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How to create novel
variation1. Mutationa. Randomb. Directed
2. Recombination
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Mutations occur at low frequencies and are mostly deleterious.
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Directed mutations are useful if we know a priori which sequence will accomplish which task.
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Recombination produces a lot of functional variation.
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Willem P. StemmerWillem P. Stemmer
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The Protocol… The Protocol…
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1. Identify a product 1. Identify a product that can be improved.that can be improved.
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… … and sold and sold with no with no controversy.controversy.
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Laundry detergents contain Laundry detergents contain the following active enzymes: the following active enzymes:
Protease — removal of Protease — removal of protein stainsprotein stains
Amylase — removal of Amylase — removal of starchy stainsstarchy stains
Lipase — removal of greasy Lipase — removal of greasy stainsstains
Peroxidase — bleachingPeroxidase — bleachingCellulase — softeningCellulase — softening
Laundry detergents contain Laundry detergents contain the following active enzymes: the following active enzymes:
Protease — removal of Protease — removal of protein stainsprotein stains
Amylase — removal of Amylase — removal of starchy stainsstarchy stains
Lipase — removal of greasy Lipase — removal of greasy stainsstains
Peroxidase — bleachingPeroxidase — bleachingCellulase — softeningCellulase — softening
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2. Select a gene that may 2. Select a gene that may improve the product.improve the product.
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3. Obtain homologous 3. Obtain homologous genes from diverse genes from diverse sources.sources.
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4. Mix “parental” genes in a solution.4. Mix “parental” genes in a solution.
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5. Fragment the genes in a 5. Fragment the genes in a number of different ways.number of different ways.
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6. Heat the solution so the 6. Heat the solution so the fragmentsfragments become single stranded. become single stranded.
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7. Cool the solution so that the 7. Cool the solution so that the gene fragments reanneal at sites of gene fragments reanneal at sites of complementarity, thus, creating complementarity, thus, creating novel recombinations. novel recombinations.
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8. The novel recombinations are 8. The novel recombinations are extended, so that double-extended, so that double-stranded heteroduplex DNA stranded heteroduplex DNA molecules are created.molecules are created.
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8. The recombination process is 8. The recombination process is repeated… repeated…
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9. … until full-length double-9. … until full-length double-stranded heteroduplex DNA stranded heteroduplex DNA molecules are created. molecules are created.
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10. The result is a library of 10. The result is a library of novel full-length genes which novel full-length genes which have different combinations of have different combinations of characteristics from the characteristics from the “parental” genes. “parental” genes.
etcetc… …
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11. Test each recombinant for the 11. Test each recombinant for the desired property.desired property.
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