Directed evolution
Encyclopedia
Directed evolution is a method used in protein engineering
to harness the power of natural selection
to evolve proteins or RNA
with desirable properties not found in nature.
Together, these three steps are termed a "round" of directed evolution. Most experiments will perform more than one round. In these experiments, the "winners" of the previous round are diversified in the next round to create a new library. At the end of the experiment, all evolved protein or RNA mutants are characterized using biochemical methods.
Performing multiple rounds of evolution is useful not only because a new library of mutants is created in each round, but because each new library uses better mutants as templates. The experiment is analogous to climbing a hill on a landscape where elevation is a function of the desired property. The goal is to reach the summit, which represents the best mutant. Each round of selection samples mutants on all sides of the starting template and selects the mutant with the highest elevation, thereby climbing the hill. A new round samples mutants on all sides of this new template and picks the highest of these, and so on until the summit is reached.
of site-directed mutagenesis
based on X-ray crystallography
data.
). It is thus possible to use this method to optimize properties that were not selected for in the original organism. This may include catalytic activity, catalytic specificity, thermostability
and many others.
Protein engineering
Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles....
to harness the power of natural selection
Natural selection
Natural selection is the nonrandom process by which biologic traits become either more or less common in a population as a function of differential reproduction of their bearers. It is a key mechanism of evolution....
to evolve proteins or RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
with desirable properties not found in nature.
A typical experiment
A typical directed evolution experiment involves three steps:- Diversification: The gene encoding the protein of interest is mutated and/or recombined at random to create a large library of gene variants. Techniques commonly used in this step are error-prone PCR and DNA shufflingDNA shufflingDNA shuffling is a way to rapidly propagate beneficial mutations in a directed evolution experiment. It is used to rapidly increase DNA library size. -Procedure:DNAse I is first used to fragment a set of parent genes into pieces of 50-100 bp in length...
. - Selection: The library is tested for the presence of mutantsMutantIn biology and especially genetics, a mutant is an individual, organism, or new genetic character, arising or resulting from an instance of mutation, which is a base-pair sequence change within the DNA of a gene or chromosome of an organism resulting in the creation of a new character or trait not...
(variants) possessing the desired property using a screen or selection. Screens enable the researcher to identify and isolate high-performing mutants by hand, while selections automatically eliminate all nonfunctional mutants. - Amplification: The variants identified in the selection or screen are replicated manyfold, enabling researchers to sequence their DNADNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
in order to understand what mutations have occurred.
Together, these three steps are termed a "round" of directed evolution. Most experiments will perform more than one round. In these experiments, the "winners" of the previous round are diversified in the next round to create a new library. At the end of the experiment, all evolved protein or RNA mutants are characterized using biochemical methods.
Likelihood of success
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.Performing multiple rounds of evolution is useful not only because a new library of mutants is created in each round, but because each new library uses better mutants as templates. The experiment is analogous to climbing a hill on a landscape where elevation is a function of the desired property. The goal is to reach the summit, which represents the best mutant. Each round of selection samples mutants on all sides of the starting template and selects the mutant with the highest elevation, thereby climbing the hill. A new round samples mutants on all sides of this new template and picks the highest of these, and so on until the summit is reached.
In vivo and in vitro
Directed evolution can be performed in living cells (in vivo evolution) or may not involve cells at all (in vitro evolution). In vivo evolution 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, but in vitro evolution is often more versatile in the types of selections that can be performed. Furthermore, in vitro evolution experiments can generate larger libraries because the library DNA need not be inserted into cells, the currently limiting step.Advantages
The advantage of the directed evolution approach is that the researcher need not understand the mechanism of the desired activity in order to improve it. An alternative method is rational designProtein design
Protein design is the design of new protein molecules, either from scratch or by making calculated variations on a known structure. The use of rational design techniques for proteins is a major aspect of protein engineering....
of site-directed mutagenesis
Site-directed mutagenesis
Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule. In general, this form of mutagenesis requires that the wild type gene sequence be known...
based on X-ray crystallography
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
data.
Uses
Most directed evolution projects seek to evolve properties that are useful to humans in an agricultural, medical or industrial context (biocatalysisBiocatalysis
Biocatalysis is the use of natural catalysts, such as protein enzymes, to perform chemical transformations on organic compounds. Both enzymes that have been more or less isolated and enzymes still residing inside living cells are employed for this task....
). It is thus possible to use this method to optimize properties that were not selected for in the original organism. This may include catalytic activity, catalytic specificity, thermostability
Thermostability
Thermostability is the quality of a substance to resist irreversible change in its chemical or physical structure at a high relative temperature.Thermostable materials may be used industrially as fire retardants...
and many others.
See also
- SCOPE (protein engineering)SCOPE (protein engineering)Structure-Based Combinatorial Protein Engineering is a synthetic biology technique for creating gene libraries of defined composition designed from structural and probabilistic constraints of the encoded proteins...
- Expanded genetic codeExpanded genetic codeAn expanded genetic code refers to an artificially modified genetic code in which one or more specific codons have been allocated to encode an amino acid which is not among the twenty/twenty-two found in nature.-Background:...
- protein methodsProtein methodsProtein methods are the techniques used to study proteins.There are genetic methods for studying proteins, methods for detecting proteins, methods for isolating and purifying proteins and other methods for characterizing the structure and function of proteins, often requiring that the protein first...
and nucleic acid methodsNucleic acid methodsNucleic acid methods are the techniques used to study nucleic acids .Purification*Phenol-chloroform extraction*minicolumn purification*RNA extractionQuantification*Abundance in weight: spectroscopic quantification... - FIND TechnologyFIND TechnologyFIND® technology is a directed evolution technology that uses DNA recombination to improve properties of proteins. It eliminates unimportant and deleterious mutations while maintaining and combining beneficial mutations that would enhance protein function....
External links
- The Ulrich Schwaneberg Research Group
- The Frances H. Arnold Research Group
- The Huimin ZhaoHuimin ZhaoDr. Huimin Zhao is a Professor of Chemical and Biomolecular Engineering at the University of Illinois, Urbana-Champaign. His research focuses on directed evolution, metabolic engineering, bioinformatics and high throughput technologies....
Research Group - The Manfred T. Reetz Group
- The Donald Hilvert Group
- Darren Hart's Group
- The David Liu Group
- The Paul A. Dalby Research Group
- DNA2.0 Protein Engineering
- SeSaM-Biotech - Directed Evolution
- GENEART - Directed Evolution: Tools for Protein Engineering
- Codexis - Platform Technology
- DIREVO - Platform Technology
- Prof. Reetz explains the principle of Directed Evolution