|Method||Genotype–phenotype linkage||Reporter, mechanism of enrichment and detection method*||Library size||Evolvable phenotypes||Advantages||Disadvantages|
*Reporter for screens or mechanism of enrichment for selections. CPR, compartmentalized partnered replication; CSR, compartmentalized self-replication; E. coli, Escherichia coli; FACS, fluorescence-activated cell sorting; HPLC, high-performance liquid chromatography; IVC, in vitro compartmentalization; MS, mass spectrometry; NMR, nuclear magnetic resonance; PACE, phage-assisted continuous directed evolution; S. cerevisiae, Saccharomyces cerevisiae.
|Colonies on solid media||Spatial separation of variants||Visible signal from fluorescent or colorimetric gene product, or surrogate substrate; detected by manual inspection||Throughput limit: 102–104||Fluorescent proteins, binding interactions and a limited number of catalytic enzymes||Straightforward implementation||Low throughput and laborious; qualitative or semi-quantitative measurements|
|Isolated liquid cultures||Spatial separation of variants||Biochemical assays, visible signal from fluorescent gene product or surrogate substrate; detected by HPLC, NMR, MS or microplate reader||Throughput limit: 102–104||Fluorescent proteins and catalytic enzymes||Straightforward implementation; flexibility in choice of reporter and detection method; quantitative measurements; many classes of enzymes and proteins can be evolved||Low throughput and laborious|
|Cell surface display||Compartmentalized (cell membrane)||Non-diffusing fluorescent reporters (fluorescent-labelled antibodies or tags); detected by FACS||Throughput limit: 108 (Refs 70,71,77)||Binding interactions, bond formation, bond cleavage and protein stability||High throughput; yeast display offers eukaryotic gene expression with chaperones and post-translational modifications||Limited applications to catalytic enzyme evolution (for example, transpeptidases77, proteases85, esterases136 and peroxidases137)|
|IVC||Compartmentalized (water–oil emulsions or polyelectrolyte shells)||Fluorescent gene product or fluorogenic substrate; detected by FACS||Throughput limit: 107–108 (Refs 70,87,88)||Fluorescent proteins, catalytic enzymes for which a fluorogenic substrate can be made||High-throughput quantitative measurements||IVC and microfluidic techniques require expertise; components for emulsion and polyelectrolyte shells must be optimized for compatibility with a given fluorogenic reporter|
|Phage, mRNA, ribosome and cell surface display||Covalent||Binding to immobilized targets||Transformation limit: 1010 (phage display)93; throughput limit: 1012–1013 (mRNA and ribosome display)97, 98||Binding interactions and a limited number of selections for catalysis99, 100, 138||Protocols for binding selection and affinity maturation are well established||Bacterial and in vitro expression biases; many eukaryotic proteins will not fold properly; not useful for evolving most enzymatic activities|
|Organismal fitness and auxotroph complementation||Compartmentalized (cell membrane)||Host fitness depends on evolving gene||Transformation limit: 108 (S. cerevisiae)95; 1010 (E. coli)96||Metabolic genes, selectable markers (antibiotic resistance) and suppressors of toxic genes||Conceptually simple||Strain optimizations can confound isolation of beneficial mutations in the evolving gene|
|IVC||Compartmentalized (water–oil emulsions)||Water–oil emulsions compartmentalize DNA, and in vitro transcription and translation yield proteins; selections for enzymes that act on their encoding DNA are common||Emulsion efficiency: 1010 (Ref. 86)||Nucleases, DNase inhibitors, DNA ligases and binding interactions||Large library sizes; lacks transformation bottleneck||Heavily geared towards evolution of enzymes with DNA substrates; IVC requires technical expertise|
|CSR||Compartmentalized (water–oil emulsions)||DNA or RNA polymerases must amplify their encoding gene in an emulsion PCR||Emulsion efficiency: 1010 (Ref. 86)||DNA and RNA polymerases||Large library sizes; lacks transformation bottleneck||By definition, this technique only applies to polymerases; IVC requires technical expertise|
|CPR||Compartmentalized (water–oil emulsions)||Emulsion PCR best amplifies genes that triggered the most Taq polymerase||Transformation limit and emulsion efficiency: 106–108 (E. coli)96, 113||RNA polymerase and tRNA synthetase (hypothetically, anything that can be linked to gene expression)||Generalizable to many proteins||Challenging to design genetic circuits that link enzymatic activity to Taq expression; IVC requires technical expertise|
|PACE||Compartmentalized (cell membrane)||Expression of essential phage gene (such as gene III) is triggered by the desired phenotype; thus, infectious progeny are made in proportion to desired activity||Phage titre limit: 108–1012 (Ref. 122)||RNA polymerase and protease (hypothetically, anything that can be linked to gene expression)||Generalizable to many proteins; continuous format enables ~100-fold more rounds of protein evolution per unit time than traditional methods||Challenging to design genetic circuits that link enzymatic activity to gene III expression; PACE requires technical expertise|
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
- Michael S. Packer &
- David R. Liu
Competing interests statement
The authors declare no competing interests.
Michael S. Packer
Michael S. Packer is a US National Science Foundation Graduate Research Fellow and a Ph.D. student in the Biophysics Program at Harvard University, Cambridge, Massachusetts, USA. Under David R. Liu's mentorship, he is working on the phage-assisted continuous evolution of proteases with altered substrate specificities.
David R. Liu
David R. Liu is Professor of Chemistry and Chemical Biology at Harvard University, Cambridge, Massachusetts, USA, and a Howard Hughes Medical Institute (HHMI) Investigator. He graduated with first place in his class at Harvard University in 1994 before entering the Ph.D. program at University of California Berkeley, USA. He earned his Ph.D. in 1999 and became Assistant Professor of Chemistry and Chemical Biology at Harvard University in the same year. He was promoted to Associate Professor in 2003 and to Full Professor in 2005. He became a HHMI Investigator in 2005 and joined the JASON Advisory Group in 2009. His research integrates chemistry and laboratory evolution to illuminate biology and enable next-generation therapeutics. He has published more than 125 papers in areas including the evolution and intracellular delivery of proteins; the characterization and engineering of genome-editing agents; and the discovery of bioactive synthetic molecules and synthetic polymers through DNA-templated synthesis, a technique developed in his laboratory. David R. Liu's homepage.
- Natural selection
A process by which individuals with the highest reproductive fitness pass on their genetic material to their offspring, thus maintaining and enriching heritable traits that are adaptive to the natural environment.
- Artificial selection
(Also known as selective breeding). A process by which human intervention in the reproductive cycle imposes a selection pressure for phenotypic traits desired by the breeder.
Diverse populations of DNA fragments that are subject to downstream screening and selection.
- Library size
The number variants that are subjected to screening and selection. Library sizes are limited by molecular cloning protocols and/or by host transformation efficiency.
- Focused mutagenesis
A strategy of diversification that introduces mutations at DNA regions expected to influence protein activity.
- Random mutagenesis
A strategy of diversification that introduces mutations in an unbiased manner throughout the entire gene.
- Mutational spectrum
The frequency of each specific type of transition and transversion. The evenness of this spectrum allows more thorough sampling of sequence space.
The process by which a cell directly acquires a foreign DNA molecule. A number of protocols allow high-efficiency transformation of microorganisms through treatments with ionic buffers, heat shock or electroporation.
- Neutral drift
A process that occurs in the presence of a purifying selection pressure to eliminate deleterious mutations. This is in contrast to genetic drift, a process by which mutations fluctuate in frequency in the absence of selection pressure.
- Degenerate codons
Codons constructed with a mixed population of nucleotides at a given position, thus sampling all possible amino acids within the constructed libraries. The most popular examples are NNK and NNS (where N can be any of the four nucleotides, K can be G or T, and S can be G or C).
- Epistatic interactions
Non-additive effects between mutations (for example, mutational synergy or synthetic lethality). As a result, the sequential acquisition of mutations is not always equivalent to mutational co-occurrence.
- Homologous recombination
A process by which separate pieces of DNA swap genetic material, guided by the annealing of complementary DNA fragments.
- Passenger mutations
(Also known as hitchhiker mutations). Unnecessary mutations that are enriched in a population owing to co-occurrence with a highly beneficial linked mutation.
The process by which a viral vector delivers a foreign DNA molecule to a cellular host.
- Evolutionary potential
The capacity of a protein to take on new functions through evolution. High thermostability allows for necessary but destabilizing mutations, and functional diversity of homologues is a demonstration of previous evolution in nature.
- Surrogate substrates
Substrate analogues that are permissive of enzymatic conversion but that, upon catalysis, exhibit chemical rearrangements that lead to altered optical properties, including visible colour, relief of fluorophore quenching, shifted fluorophore excitation or emission, and downstream chemiluminescence.
- Fluorescence-activated cell sorting
(FACS). A flow cytometry method in which an aqueous suspension of cells or cell-like compartments is measured for fluorescence (often at multiple wavelengths) one cell at a time and subsequently separated based on a fluorescence threshold.
- Negative screen
A screening method that involves depletion of an undesired phenotype.
- Positive screening
Enrichment for a desired activity such as improved kinetics, tolerance to unnatural conditions and acceptance of new substrates.
- Transformation bottleneck
The efficiency at which DNA library members are transferred into the host organism, thus restricting the number of variants that can be assessed by in vivo selection and screening.
- Auxotroph complementation
The ability of functional library members to resolve a metabolic defect in the host, leading to replication of DNA that encodes active library members.