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Directed evolution

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</style><div role="note" class="hatnote navigation-not-searchable">Not to be confused with <a href="Directed_evolution_(transhumanism)" title="Directed evolution (transhumanism)">Directed evolution (transhumanism)</a>.</div>

Directed evolution (DE) is a method used in <a href="Protein_engineering" title="Protein engineering">protein engineering</a> that mimics the process of <a href="Natural_selection" title="Natural selection">natural selection</a> to steer <a href="Proteins" class="mw-redirect" title="Proteins">proteins</a> or <a href="Nucleic_acid" title="Nucleic acid">nucleic acids</a> toward a user-defined goal.<a href="#cite_note-ReferenceA-1">[1]</a> It consists of subjecting a <a href="Gene" title="Gene">gene</a> to iterative rounds of <a href="Mutagenesis" title="Mutagenesis">mutagenesis</a> (creating a library of variants), selection (expressing those variants and isolating members with the desired function) and amplification (generating a template for the next round). It can be performed <a href="In_vivo" title="In vivo">in vivo</a> (in living organisms), or <a href="In_vitro" title="In vitro">in vitro</a> (in cells or free in solution). Directed evolution is used both for <a href="Protein_engineering" title="Protein engineering">protein engineering</a> as an alternative to <a href="Rational_design" title="Rational design">rationally designing</a> modified proteins, as well as for <a href="Experimental_evolution" title="Experimental evolution">experimental evolution</a> studies of fundamental <a href="Evolution" title="Evolution">evolutionary principles</a> in a controlled, laboratory environment.

<div class="mw-heading mw-heading2"><h2 id="History">History</h2></div>

Directed evolution has its origins in the 1960s<a href="#cite_note-:0-2">[2]</a> with the evolution of <a href="RNA" title="RNA">RNA molecules</a> in the "<a href="Spiegelman's_Monster" title="Spiegelman's Monster">Spiegelman's Monster</a>" experiment.<a href="#cite_note-An_extracellular_Darwinian_experime-3">[3]</a> The concept was extended to protein evolution via evolution of bacteria under selection pressures that favoured the evolution of a single gene in its genome.<a href="#cite_note-4">[4]</a>

Early <a href="Phage_display" title="Phage display">phage display</a> techniques in the 1980s allowed targeting of mutations and selection to a single protein.<a href="#cite_note-5">[5]</a> This enabled selection of enhanced <a href="Binding_protein" title="Binding protein">binding proteins</a>, but was not yet compatible with selection for catalytic activity of <a href="Enzyme" title="Enzyme">enzymes</a>.<a href="#cite_note-6">[6]</a> Methods to evolve enzymes were developed in the 1990s and brought the technique to a wider scientific audience.<a href="#cite_note-7">[7]</a> The field rapidly expanded with new methods for making libraries of gene variants and for screening their activity.<a href="#cite_note-:0-2">[2]</a><a href="#cite_note-8">[8]</a> The development of directed evolution methods was honored in 2018 with the awarding of the <a href="Nobel_Prize_in_Chemistry" title="Nobel Prize in Chemistry">Nobel Prize in Chemistry</a> to <a href="Frances_Arnold" title="Frances Arnold">Frances Arnold</a> for evolution of enzymes, and <a href="George_Smith_(chemist)" title="George Smith (chemist)">George Smith</a> and <a href="Gregory_Winter" title="Gregory Winter">Gregory Winter</a> for phage display.<a href="#cite_note-9">[9]</a>

<div class="mw-heading mw-heading2"><h2 id="Principles">Principles</h2></div>

Directed evolution is a mimic of the natural evolution cycle in a laboratory setting. Evolution requires three things to happen: <a href="Genetic_diversity" title="Genetic diversity">variation</a> between replicators, that the variation causes <a href="Fitness_(biology)" title="Fitness (biology)">fitness differences</a> upon which selection acts, and that this variation is <a href="Heritability" title="Heritability">heritable</a>. In DE, a single gene is evolved by iterative rounds of mutagenesis, selection or screening, and amplification.<a href="#cite_note-10">[10]</a> Rounds of these steps are typically repeated, using the best variant from one round as the template for the next to achieve stepwise improvements.

The likelihood of success in a directed evolution experiment is directly related to the total library size, as evaluating more mutants increases the chances of finding one with the desired properties.<a href="#cite_note-11">[11]</a>

<div class="mw-heading mw-heading3"><h3 id="Generating_variation">Generating variation</h3></div>

The first step in performing a cycle of directed evolution is the generation of a library of variant genes. The <a href="Sequence_space" title="Sequence space">sequence space</a> for random sequence is vast (10130 possible sequences for a 100 <a href="Amino_acid" title="Amino acid">amino acid</a> protein) and extremely sparsely populated by functional proteins. Neither experimental,<a href="#cite_note-Lipovsek_D_2004-12">[12]</a> nor natural<a href="#cite_note-Dryden_DT_2008-13">[13]</a> evolution can ever get close to sampling so many sequences. Of course, natural evolution samples variant sequences close to functional protein sequences and this is imitated in DE by mutagenising an already functional gene.

Some calculations suggest it is entirely feasible that for all practical (i.e. functional and structural) purposes, protein sequence space has been fully explored during the course of evolution of life on Earth.<a href="#cite_note-Dryden_DT_2008-13">[13]</a>

The starting gene can be mutagenised by random <a href="Point_mutations" class="mw-redirect" title="Point mutations">point mutations</a> (by chemical mutagens or error prone <a href="Polymerase_chain_reaction" title="Polymerase chain reaction">PCR</a>)<a href="#cite_note-14">[14]</a><a href="#cite_note-15">[15]</a> and <a href="Indel" title="Indel">insertions and deletions</a> (by transposons).<a href="#cite_note-16">[16]</a> <a href="Genetic_recombination" title="Genetic recombination">Gene recombination</a> can be mimicked by <a href="DNA_shuffling" title="DNA shuffling">DNA shuffling</a><a href="#cite_note-17">[17]</a><a href="#cite_note-18">[18]</a> of several sequences (usually of more than 70% sequence identity) to jump into regions of sequence space between the shuffled parent genes. Finally, specific regions of a gene can be systematically randomised<a href="#cite_note-19">[19]</a> for a more focused approach based on structure and function knowledge. Depending on the method, the library generated will vary in the <a href="Distribution_of_fitness_effects" class="mw-redirect" title="Distribution of fitness effects">proportion of functional variants</a> it contains. Even if an organism is used to express the gene of interest, by mutagenising only that gene the rest of the organism's genome remains the same and can be ignored for the evolution experiment (to the extent of providing a constant genetic environment).

<div class="mw-heading mw-heading3"><h3 id="Detecting_fitness_differences">Detecting fitness differences</h3></div>

The majority of <a href="Mutation" title="Mutation">mutations</a> are deleterious and so libraries of mutants tend to mostly have variants with reduced <a href="Catalytic_activity" class="mw-redirect" title="Catalytic activity">activity</a>.<a href="#cite_note-20">[20]</a> Therefore, a high-throughput <a href="Assay" title="Assay">assay</a> is vital for measuring activity to find the rare variants with beneficial mutations that improve the desired properties. Two main categories of method exist for isolating functional variants. Selection systems directly couple protein function to survival of the gene, whereas screening systems individually assay each variant and allow a quantitative threshold to be set for sorting a variant or population of variants of a desired activity. Both selection and screening can be performed in living cells (in vivo evolution) or performed directly on the <a href="Protein" title="Protein">protein</a> or <a href="RNA" title="RNA">RNA</a> without any cells (in vitro evolution).<a href="#cite_note-21">[21]</a><a href="#cite_note-22">[22]</a>

During in vivo evolution, each cell (usually <a href="Bacteria" title="Bacteria">bacteria</a> or <a href="Yeast" title="Yeast">yeast</a>) is <a href="Transformation_(genetics)" class="mw-redirect" title="Transformation (genetics)">transformed</a> with a <a href="Plasmid" title="Plasmid">plasmid</a> containing a different member of the variant library. In this way, only the gene of interest differs between the cells, with all other genes being kept the same. The cells express the protein either in their <a href="Cytoplasm" title="Cytoplasm">cytoplasm</a> or <a href="Plasma_membrane" class="mw-redirect" title="Plasma membrane">surface</a> where its function can be tested. This format has the advantage of selecting for properties in a cellular environment, which is useful when the evolved protein or RNA is to be used in living organisms. When performed without cells, DE involves using <a href="In_vitro_compartmentalization#In_Vitro_transcription.2Ftranslation" title="In vitro compartmentalization">in vitro transcription translation</a> to produce proteins or RNA free in solution or compartmentalised in <a href="In_vitro_compartmentalization" title="In vitro compartmentalization">artificial microdroplets</a>. This method has the benefits of being more versatile in the selection conditions (e.g. temperature, solvent), and can express proteins that would be toxic to cells. Furthermore, in vitro evolution experiments can generate far larger libraries (up to 1015) because the library DNA need not be <a href="Transformation_(genetics)" class="mw-redirect" title="Transformation (genetics)">inserted</a> into cells (often a limiting step).

<div class="mw-heading mw-heading4"><h4 id="Selection">Selection</h4></div>

Selection for <a href="Protein_binding" class="mw-redirect" title="Protein binding">binding activity</a> is conceptually simple. The target molecule is immobilised on a solid support, a library of variant proteins is flowed over it, poor binders are washed away, and the remaining bound variants recovered to isolate their genes.<a href="#cite_note-23">[23]</a> Binding of an enzyme to immobilised covalent <a href="Enzyme_inhibitor" title="Enzyme inhibitor">inhibitor</a> has been also used as an attempt to isolate active catalysts. This approach, however, only selects for single catalytic turnover and is not a good model of substrate binding or true substrate reactivity. If an enzyme activity can be made necessary for cell survival, either by synthesizing a vital metabolite, or destroying a toxin, then cell survival is a function of enzyme activity.<a href="#cite_note-Leemhuis_H_2005-24">[24]</a><a href="#cite_note-25">[25]</a> Such systems are generally only limited in throughput by the <a href="Transformation_(genetics)" class="mw-redirect" title="Transformation (genetics)">transformation</a> efficiency of cells. They are also less expensive and labour-intensive than screening, however they are typically difficult to engineer, prone to artefacts and give no information on the <a href="Distribution_of_fitness_effects" class="mw-redirect" title="Distribution of fitness effects">range of activities</a> present in the library.

<div class="mw-heading mw-heading4"><h4 id="Screening">Screening</h4></div>

An alternative to selection is a screening system. Each variant gene is individually <a href="Protein_expression_(biotechnology)" class="mw-redirect" title="Protein expression (biotechnology)">expressed</a> and <a href="Assay" title="Assay">assayed</a> to quantitatively measure the activity (most often by a <a href="Colorimetry" title="Colorimetry">colourgenic</a> or <a href="Fluorogenic" title="Fluorogenic">fluorogenic</a> product). The variants are then ranked and the experimenter decides which variants to use as templates for the next round of DE. Even the most high throughput assays usually have lower coverage than selection methods but give the advantage of producing detailed information on each one of the screened variants. This disaggregated data can also be used to characterise the distribution of activities in libraries which is not possible in simple selection systems. Screening systems, therefore, have advantages when it comes to experimentally characterising adaptive evolution and fitness landscapes.

<div class="mw-heading mw-heading3"><h3 id="Ensuring_heredity">Ensuring heredity</h3></div>

When functional proteins have been isolated, it is necessary that their genes are too, therefore a <a href="Heredity" title="Heredity">genotype–phenotype</a> link is required.<a href="#cite_note-Leemhuis_H_2005-24">[24]</a> This can be covalent, such as <a href="MRNA_display" title="MRNA display">mRNA display</a> where the <a href="MRNA" class="mw-redirect" title="MRNA">mRNA</a> gene is linked to the protein at the end of translation by puromycin.<a href="#cite_note-Lipovsek_D_2004-12">[12]</a> Alternatively the protein and its gene can be co-localised by compartmentalisation in living cells<a href="#cite_note-26">[26]</a> or emulsion droplets.<a href="#cite_note-27">[27]</a> The gene sequences isolated are then amplified by PCR or by transformed host bacteria. Either the single best sequence, or a pool of sequences can be used as the template for the next round of mutagenesis. The repeated cycles of Diversification-Selection-Amplification generate protein variants adapted to the applied selection pressures.

<div class="mw-heading mw-heading2"><h2 id="Comparison_to_rational_protein_design">Comparison to rational protein design</h2></div>

<div class="mw-heading mw-heading3"><h3 id="Advantages_of_directed_evolution">Advantages of directed evolution</h3></div>

<a href="Protein_design" title="Protein design">Rational design</a> of a protein relies on an in-depth knowledge of the <a href="Protein_structure" title="Protein structure">protein structure</a>, as well as its <a href="Catalytic_mechanism" class="mw-redirect" title="Catalytic mechanism">catalytic mechanism</a>.<a href="#cite_note-28">[28]</a><a href="#cite_note-29">[29]</a> Specific changes are then made by <a href="Site-directed_mutagenesis" title="Site-directed mutagenesis">site-directed mutagenesis</a> in an attempt to change the function of the protein. A drawback of this is that even when the structure and mechanism of action of the protein are well known, the change due to mutation is still difficult to predict. Therefore, an advantage of DE is that there is no need to understand the mechanism of the desired activity or how mutations would affect it.<a href="#cite_note-ReferenceB-30">[30]</a>

<div class="mw-heading mw-heading3"><h3 id="Limitations_of_directed_evolution">Limitations of directed evolution</h3></div>

A restriction of directed evolution is that a high-throughput assay is required in order to measure the effects of a large number of different random mutations. This can require extensive research and development before it can be used for directed evolution. Additionally, such assays are often highly specific to monitoring a particular activity and so are not transferable to new DE experiments.<a href="#cite_note-31">[31]</a>

Additionally, selecting for improvement in the assayed function simply generates improvements in the assayed function. To understand how these improvements are achieved, the properties of the evolving enzyme have to be measured. Improvement of the assayed activity can be due to improvements in enzyme catalytic activity or enzyme concentration. There is also no guarantee that improvement on one substrate will improve activity on another. This is particularly important when the desired activity cannot be directly screened or selected for and so a ‘proxy’ substrate is used. DE can lead to evolutionary specialisation to the proxy without improving the desired activity. Consequently, choosing appropriate screening or selection conditions is vital for successful DE.<a href="#cite_note-32">[32]</a>

The speed of evolution in an experiment also poses a limitation on the utility of directed evolution. For instance, evolution of a particular phenotype, while theoretically feasible, may occur on time-scales that are not practically feasible.<a href="#cite_note-33">[33]</a> Recent theoretical approaches have aimed to overcome the limitation of speed through an application of <a href="Shortcuts_to_adiabaticity" title="Shortcuts to adiabaticity">counter-diabatic driving</a> techniques from statistical physics, though this has yet to be implemented in a directed evolution experiment.<a href="#cite_note-34">[34]</a>

<div class="mw-heading mw-heading3"><h3 id="Combinatorial_approaches">Combinatorial approaches</h3></div>

Combined, 'semi-rational' approaches are being investigated to address the limitations of both rational design and directed evolution.<a href="#cite_note-ReferenceA-1">[1]</a><a href="#cite_note-Goldsmith_M_2012-35">[35]</a> Beneficial mutations are rare, so large numbers of random mutants have to be screened to find improved variants. 'Focused libraries' concentrate on randomising regions thought to be richer in beneficial mutations for the mutagenesis step of DE. A focused library contains fewer variants than a traditional random mutagenesis library and so does not require such high-throughput screening.

Creating a focused library requires some knowledge of which residues in the structure to mutate. For example, knowledge of the <a href="Active_site" title="Active site">active site</a> of an enzyme may allow just the residues known to interact with the <a href="Enzyme_substrate_(biology)" class="mw-redirect" title="Enzyme substrate (biology)">substrate</a> to be randomised.<a href="#cite_note-36">[36]</a><a href="#cite_note-37">[37]</a> Alternatively, knowledge of which protein regions are <a href="Segregating_site" title="Segregating site">variable</a> in nature can guide mutagenesis in just those regions.<a href="#cite_note-38">[38]</a><a href="#cite_note-39">[39]</a>

<div class="mw-heading mw-heading2"><h2 id="Applications">Applications</h2></div>

Directed evolution is frequently used for <a href="Protein_engineering" title="Protein engineering">protein engineering</a> as an alternative to <a href="Rational_design" title="Rational design">rational design</a>,<a href="#cite_note-ReferenceC-40">[40]</a> but can also be used to investigate fundamental questions of enzyme evolution.<a href="#cite_note-41">[41]</a>

<div class="mw-heading mw-heading3"><h3 id="Protein_engineering">Protein engineering</h3></div>

As a protein engineering tool, DE has been most successful in three areas:

<ol><li>Improving <a href="Protein_stability" class="mw-redirect" title="Protein stability">protein stability</a> for biotechnological use at high temperatures or in harsh solvents<a href="#cite_note-42">[42]</a><a href="#cite_note-43">[43]</a><a href="#cite_note-Favor_2020-44">[44]</a></li>

<li>Improving <a href="Binding_affinity" class="mw-redirect" title="Binding affinity">binding affinity</a> of <a href="Monoclonal_antibody_therapy" title="Monoclonal antibody therapy">therapeutic antibodies</a> (<a href="Affinity_maturation" title="Affinity maturation">Affinity maturation</a>)<a href="#cite_note-45">[45]</a> and the activity of de novo <a href="Protein_design" title="Protein design">designed enzymes</a><a href="#cite_note-ReferenceB-30">[30]</a></li>

<li>Altering <a href="Substrate_specificity" class="mw-redirect" title="Substrate specificity">substrate specificity</a> of existing enzymes,<a href="#cite_note-46">[46]</a><a href="#cite_note-47">[47]</a><a href="#cite_note-48">[48]</a><a href="#cite_note-49">[49]</a> (often for use in industry)<a href="#cite_note-ReferenceC-40">[40]</a></li></ol>

<div class="mw-heading mw-heading3"><h3 id="Evolution_studies">Evolution studies</h3></div>

<div role="note" class="hatnote navigation-not-searchable">See also: <a href="Experimental_evolution" title="Experimental evolution">Experimental evolution</a></div>

The study of natural <a href="Evolution" title="Evolution">evolution</a> is traditionally based on extant organisms and their genes. However, research is fundamentally limited by the lack of <a href="Fossil" title="Fossil">fossils</a> (and particularly the lack of <a href="Ancient_DNA" title="Ancient DNA">ancient DNA</a> sequences)<a href="#cite_note-50">[50]</a><a href="#cite_note-51">[51]</a> and incomplete knowledge of ancient environmental conditions. Directed evolution investigates evolution in a controlled system of genes for individual <a href="Enzymes" class="mw-redirect" title="Enzymes">enzymes</a>,<a href="#cite_note-52">[52]</a><a href="#cite_note-53">[53]</a><a href="#cite_note-Goldsmith_M_2012-35">[35]</a> <a href="Ribozymes" class="mw-redirect" title="Ribozymes">ribozymes</a><a href="#cite_note-54">[54]</a> and <a href="Replicator_(evolution_unit)" class="mw-redirect" title="Replicator (evolution unit)">replicators</a><a href="#cite_note-55">[55]</a><a href="#cite_note-An_extracellular_Darwinian_experime-3">[3]</a> (similar to experimental evolution of <a href="Eukaryotes" class="mw-redirect" title="Eukaryotes">eukaryotes</a>,<a href="#cite_note-56">[56]</a><a href="#cite_note-57">[57]</a> <a href="Prokaryotes" class="mw-redirect" title="Prokaryotes">prokaryotes</a><a href="#cite_note-58">[58]</a> and <a href="Viruses" class="mw-redirect" title="Viruses">viruses</a><a href="#cite_note-59">[59]</a>).

DE allows control of <a href="Selection_pressure" class="mw-redirect" title="Selection pressure">selection pressure</a>, <a href="Mutation_rate" title="Mutation rate">mutation rate</a> and <a href="Environment_(biophysical)" class="mw-redirect" title="Environment (biophysical)">environment</a> (both the <a href="Abiotic_component" title="Abiotic component">abiotic environment</a> such as temperature, and the biotic environment, such as other genes in the organism). Additionally, there is a complete record of all evolutionary intermediate genes. This allows for detailed measurements of evolutionary processes, for example <a href="Epistasis" title="Epistasis">epistasis</a>, <a href="Evolvability" title="Evolvability">evolvability</a>, <a href="Adaptationism#Genetic_constraints" title="Adaptationism">adaptive constraint</a><a href="#cite_note-60">[60]</a><a href="#cite_note-61">[61]</a> <a href="Fitness_landscapes" class="mw-redirect" title="Fitness landscapes">fitness landscapes</a>,<a href="#cite_note-62">[62]</a> and <a href="Neutral_networks" class="mw-redirect" title="Neutral networks">neutral networks</a>.<a href="#cite_note-63">[63]</a>

<div class="mw-heading mw-heading3"><h3 id="Adaptive_laboratory_evolution_of_microbial_proteomes">Adaptive laboratory evolution of microbial proteomes</h3></div>

The natural <a href="Amino_acid" title="Amino acid">amino acid</a> composition of <a href="Proteome" title="Proteome">proteomes</a> can be changed by global canonical amino acids substitutions with suitable noncanonical counterparts under the experimentally imposed <a href="Selective_pressure" class="mw-redirect" title="Selective pressure">selective pressure</a>. For example, global proteome-wide substitutions of natural amino acids with fluorinated analogs have been attempted in Escherichia coli<a href="#cite_note-64">[64]</a> and Bacillus subtilis.<a href="#cite_note-65">[65]</a> A complete <a href="Tryptophan" title="Tryptophan">tryptophan</a> substitution with thienopyrrole-alanine in response to 20899 UGG <a href="Codon" class="mw-redirect" title="Codon">codons</a> in Escherichia coli was reported in 2015 by <a href="Nediljko_Budisa" title="Nediljko Budisa">Budisa</a> and <a href="Dieter_S%C3%B6ll" title="Dieter Söll">Söll</a>.<a href="#cite_note-66">[66]</a> The experimental evolution of microbial strains with a clear-cut accommodation of an additional amino acid is expected to be instrumental for widening the <a href="Genetic_code" title="Genetic code">genetic code</a> experimentally.<a href="#cite_note-67">[67]</a> Directed evolution typically targets a particular gene for <a href="Mutagenesis" title="Mutagenesis">mutagenesis</a> and then screens the resulting variants for a <a href="Phenotype" title="Phenotype">phenotype</a> of interest, often independent of <a href="Fitness_(biology)" title="Fitness (biology)">fitness</a> effects, whereas adaptive laboratory evolution selects many <a href="Genome" title="Genome">genome</a>-wide mutations that contribute to the fitness of actively growing cultures.<a href="#cite_note-68">[68]</a>

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<ul><li>Applications:

<ul><li><a href="Protein_engineering" title="Protein engineering">Protein engineering</a></li>

<li><a href="Enzyme_engineering" class="mw-redirect" title="Enzyme engineering">Enzyme engineering</a></li>

<li><a href="Protein_design" title="Protein design">Protein design</a></li>

<li><a href="Expanded_genetic_code" title="Expanded genetic code">Expanded genetic code</a></li>

<li><a href="Xenobiology" title="Xenobiology">Xenobiology</a></li></ul></li>

<li>Mutagenesis:

<ul><li><a href="Random_mutagenesis" class="mw-redirect" title="Random mutagenesis">Random mutagenesis</a></li>

<li><a href="Saturated_mutagenesis" class="mw-redirect" title="Saturated mutagenesis">Saturated mutagenesis</a></li>

<li><a href="Staggered_extension_process" title="Staggered extension process">Staggered extension process</a></li></ul></li>

<li>Selection and screening:

<ul><li><a href="Yeast_display" title="Yeast display">Yeast display</a></li>

<li><a href="Bacterial_display" title="Bacterial display">Bacterial display</a></li>

<li><a href="Phage_display" title="Phage display">Phage display</a></li>

<li><a href="Ribosome_display" title="Ribosome display">Ribosome display</a></li>

<li><a href="MRNA_display" title="MRNA display">mRNA display</a></li>

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<div class="mw-heading mw-heading2"><h2 id="References">References</h2></div>

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<div class="mw-heading mw-heading2"><h2 id="External_links">External links</h2></div>

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<ul><li>Research groups

<ul><li><a rel="nofollow" class="external text" href="http://www.weizmann.ac.il/Biological_Chemistry/scientist/Tawfik/">The Dan Tawfik Research Group</a></li>

<li><a rel="nofollow" class="external text" href="http://www.biotec.rwth-aachen.de/">The Ulrich Schwaneberg Research Group</a></li>

<li><a rel="nofollow" class="external text" href="http://www.che.caltech.edu/groups/fha/">The Frances Arnold Research Group</a> <a rel="nofollow" class="external text" href="https://web.archive.org/web/20191229040335/http://www.che.caltech.edu/groups/fha/">Archived</a> 2019-12-29 at the <a href="Wayback_Machine" title="Wayback Machine">Wayback Machine</a></li>

<li><a rel="nofollow" class="external text" href="http://scs.illinois.edu/~zhaogrp/">The Huimin Zhao Research Group</a></li>

<li><a rel="nofollow" class="external text" href="https://web.archive.org/web/20090426053856/http://www.kofo.mpg.de/reetz/">The Manfred Reetz Research Group</a></li>

<li><a rel="nofollow" class="external text" href="https://web.archive.org/web/20160721032623/http://www.protein.ethz.ch/directed.html">The Donald Hilvert Group</a></li>

<li><a rel="nofollow" class="external text" href="https://web.archive.org/web/20160424073647/http://www.embl.fr/research/unit/hart/index.html">The Darren Hart Research Group</a></li>

<li><a rel="nofollow" class="external text" href="http://www.liulab.com/">The Chang Liu Research Group</a></li>

<li><a rel="nofollow" class="external text" href="http://evolve.harvard.edu/">The David Liu Research Group</a></li>

<li><a rel="nofollow" class="external text" href="http://www.cchem.berkeley.edu/clarkgrp/">The Douglas Clark Research Group</a></li>

<li><a rel="nofollow" class="external text" href="http://www.homepages.ucl.ac.uk/~ucbepad/PADframeset.htm">The Paul Dalby Research Group</a></li>

<li><a rel="nofollow" class="external text" href="http://chemsynbio.com/new/output/index.html">The Ned Budisa Research Group</a></li></ul></li>

<li><a rel="nofollow" class="external text" href="https://www.sesam-biotech.com/enzyme-services/engineering/">SeSaM-Biotech – Directed Evolution</a> <a rel="nofollow" class="external text" href="https://web.archive.org/web/20180527125308/https://www.sesam-biotech.com/enzyme-services/engineering/">Archived</a> 2018-05-27 at the <a href="Wayback_Machine" title="Wayback Machine">Wayback Machine</a></li>

<li><a rel="nofollow" class="external text" href="https://web.archive.org/web/20110718212547/http://www.chymiatrie.de/index.php/component/content/article/133-video-37">Prof. Reetz explains the principle of Directed Evolution</a></li>

<li><a rel="nofollow" class="external text" href="http://www.codexis.com/">Codexis, Inc.</a></li></ul>

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