GeneArt Directed Evolution | Thermo Fisher Scientific

Mutation, selection, reproduction—repeated for millions of generations. Every biologist is familiar with how evolution has resulted in the incredible diversity and adaptations found in the natural world. Directed evolution is a protein engineering method that involves a few rounds of iterative variant library creation and selection for desired traits. This is a powerful tool for researchers to develop proteins with enhanced or novel functions in a short period of time.

Benefits of designing variant libraries with GeneArt services

Invitrogen GeneArt Custom DNA Libraries cover a variety of different approaches for creating genetic variants with control and efficiency. Starting from a single-base pair exchange to full synthetic libraries, we offer custom solutions to create genetic diversity and proteins with the characteristics you need.

No physical template required
Generate variants that cannot be created using conventional methods
Maximum variation where you want it, and maximized sequence integrity in unmutated regions
Helps improve the likelihood of obtaining useful variants
Helps significantly reduce screening efforts compared to conventional mutagenesis methods
Include next-generation sequencing quality control for your library (optional)

Featured GeneArt directed evolution services

Combinatorial libraries

Introduce random variation in multiple codons, generating more than 10¹² variants, and customize the amino acid composition with TRIM technology.

Learn more about combinatorial library services and TRIM technology

Controlled randomization service

Introduce unbiased random mutations at the frequency you request, confining variation to selected regions or mutagenizing the entire ORF, more than 10¹¹ variants.

Learn more about our controlled randomization services

Site-saturation mutagenesis

Perform systematic mutagenesis to substitute the wild-type codon at specific positions with codons for up to all 19 alternative amino acids. Customize mutagenesis to fit your precise needs.

Learn more about our site-saturation mutagenesis services

Strings libraries

Choose from normalized HTP Strings DNA fragments in arrayed format or pools of DNA fragments containing up to three blocks of degenerate nucleotides each with randomized distribution (full IUPAC code).

Learn more about our Strings library synthesis services

Mutagenesis services

Utilize rapid, economical site-directed mutagenesis of existing DNA templates using a PCR-based approach.

Learn more about our mutagenesis services

Choosing a custom library

Use the decision tree (Figure 1) to choose the right GeneArt mutagenesis and library synthesis services.

Flowchart guiding selection of GeneArt mutagenesis and library synthesis services

Figure 1. Selection guide: GeneArt mutagenesis and library synthesis services.

Table 1. Comparison of the GeneArt mutagenesis and library synthesis services.

	GeneArt Strings DNA libraries	GeneArt site-saturation mutagenesis	GeneArt controlled randomization service	GeneArt combinatorial libraries
Advantage	Cost efficient Fast	Fewer variants to screen Fast service Cost efficient	Precise average mutation rate per construct Fast service	High diversity Control of amino acid distribution No unwanted stop codons
Design flexibility	+ Full IUPAC code available; however limited control over occurring amino acids	++ Systematic identification of beneficial amino acid substitutions	++ Random nucleotides within a defined region will be mutated at a defined average mutation rate	+++ TRIM technology allows for the accurate determination of amino acid ratios at each position
Correctness	+ Gene synthesis process used, more unintended mutations occur	+++ Error-free template enables best possible correctness of results	++ Low ancillary mutation rate, fewer silent mutations	+++ Error-free template enables superior correctness of results
Complexity	<10¹¹	16–19 non-wild type amino acids per codon position	>10¹⁰–10¹¹	>10¹¹ >10¹² optional
Cost	$	$	$$	$$–$$$
Production time	+++ 10–15 business days	++	+	+ 4–6 weeks
Quality control	Pool sequenced Size selection	Pool sequenced Sequencing of a sampling of individual transformants	Pool sequenced Sequencing of a sampling of individual transformants NGS option available	Pool sequenced Sequencing of a sampling of individual transformants NGS option available

Synthetic variant libraries

Synthetic variant libraries are designed to overcome some of the limitations associated with conventional mutagenesis and DNA shuffling. These libraries use advanced techniques to create a more focused and comprehensive collection of genetic variants. By precisely controlling the introduction of mutations, variant libraries can systematically explore a wider range of sequence space. This targeted approach increases the likelihood of identifying beneficial mutations and reduces the presence of non-functional variants. Additionally, variant libraries can be tailored to include specific types of mutations, such as those affecting protein function or stability, thereby enhancing the efficiency and effectiveness of the screening process.

Circular diagram of gene evolution: mutation of natural gene, protein expression, selection, and amplification to improve the gene

Click image to enlarge

Figure 2. Directed evolution

Drawbacks of conventional mutagenesis

Position and nature of mutations cannot be controlled
Mutant libraries contain many silent mutations and ill-placed stop codons
Only a tiny fraction of all possible sequence variants can be generated
Finding the best possible variant is extremely unlikely

Drawbacks of DNA shuffling

Requires stretches of homologous sequences for high-efficiency recombination sites
Needs DNA as starting material
Traditional DNA shuffling typically does not separate and recombine adjacent single-nucleotide polymorphisms
Single-nucleotide polymorphisms have the potential to result in useful protein variants

Why choose GeneArt custom variant libraries for directed evolution

GeneArt DNA libraries for protein engineering address many of the challenges associated with conventional variant-library construction techniques. De novo gene synthesis enables construction of virtually any gene variation so that your library encodes maximum variability. Supported by the GeneArt GeneOptimizer algorithm for sequence design, synthetic libraries achieve thorough representation of desired variants with the specified distribution of nucleotides in areas targeted for partial degeneracy or full randomization. The GeneArt Gene Synthesis process simultaneously minimizes the introduction of unwanted mutants and erroneous changes to unmutated portions of your constructs. This approach dramatically reduces the number of variants—economizing screening time, reagents, and effort—while helping to increase your chance of success. Because library synthesis is largely automated, we can offer rapid production times so that you can get started quickly. Our rigorous quality control systems include sequencing (and optional next-generation sequencing), statistical sequence analysis, and real-time PCR diversity analysis (control procedures are tailored to individual product lines).

Tell us about your project to receive information on pricing and production time.

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Gene synthesis and cloning services

Strings DNA fragments synthesis service

Protein expression and purification services

GeneArt directed evolution FAQs

What are the IUPAC nucleotide ambiguity codes?

IUPAC nucleotide code	Base
A	Adenine
C	Cytosine
G	Guanine
T (or U)	Thymine (or Uracil)
R	A or G
Y	C or T
S	G or C
W	A or T
K	G or T
M	A or C
B	C or G or T
D	A or G or T
H	A or C or T
V	A or C or G
N	Any base

What should a degenerate library look like for the best chance of finding an improved version of my protein?

The answer depends on the specifics of your project. In general, it is advantageous to keep the diversity of a library as low as possible, targeting only the regions of a gene/protein that are likely to be functionally important. The following information can help determine this: crystal structure, conserved motifs, presence of homologs, etc. Feel free to contact us and benefit from our vast experience.

What are the advantages of synthetic libraries over conventional protocols for creating diversity?

Conventional protocols for degenerate library creation (e.g., error-prone PCR) incorporate many unwanted mutations. Moreover, methods like DNA shuffling cannot typically cause recombination of directly adjacent mutations. Synthetic combinatorial libraries, on the other hand, limit the introduction of mutations to defined regions at the precise frequencies requested. In addition, adjacent mutations will be recombined (shuffled) independent of their proximity.

What are the advantages of trinucleotide mutagenesis (TRIM) technology over using ambiguous nucleotides for creating diversity?

The use of trinucleotide mutagenesis (TRIM) technology to create diversity enables the replacement of complete codons instead of altering single nucleotides. You thus have complete control over which amino acids appear in each position and in what ratios, while at the same time avoiding the undesired introduction of stop codons. Because complete codons are replaced, TRIM technology also results in fewer out-of-frame mutations than other mutagenesis technologies.

What codons are available for creating diversity in TRIM libraries?

We routinely use optimized codons for the most common expression hosts (human, CHO, and E. coli). All other codons are available upon request. Contact us for details.

Why do I need rational diversity when I can screen a large number of random mutants anyway?

There are several reasons:

Because you already know that certain amino acid substitutions disturb the function of your protein (e.g., cysteines in complementarity determining regions (CDRs))
Because the number of possible variants of a protein is astronomically high, exceeding the capacity of even the highest-throughput screening capabilities by many orders of magnitude. The fewer useless mutations—such as those occurring in less important regions of the protein or that cause frame shifts or stop codons—the better your chances of finding a variant that results in the desired phenotype
Some screening assays are cost- and labor-intensive; thus, screening fewer clones can save you time and money

What types of degenerated libraries are available?

Combinatorial libraries (up to 10¹¹ variants)—simultaneous randomization of multiple codons, TRIM technology optional
Site-saturation mutagenesis—randomization of a single codon with every possible non–wild type variant at different positions
Controlled randomization (up to 10¹¹ variants)—unbiased random substitutions with defined frequency
GeneArt Strings DNA libraries—linear DNA fragments that can contain up to three regions of randomized nucleotides

What if my library design doesn’t fit any of these library types?

If you require a degenerate library with an unusual design, please contact us. We will consider any project, and in most cases, we can find a solution to fulfill your requirements.

Is it possible to introduce diversity with nucleotide mixes other than the fixed ratios defined by ambiguous nucleotides?

Yes. GeneArt Combinatorial Libraries can be randomized using any nucleotide mix you require, down to single-digit percentages of given nucleotides.

What material do I have to supply for degenerate library synthesis?

No material at all. All we need for library creation is the sequence file, submitted electronically, and information about the position and nature of the sites you want to randomize. We can provide a quote for your project and, if needed, you can then provide detailed information about your library request to our production scientists prior to starting the project.

Can a library be subcloned in my vector?

Yes. We can subclone your gene into any commercially available Invitrogen vector or into a custom vector that you provide.

How are libraries supplied?

Two standard options are available:

As a set of linear DNA fragments, ready for restriction enzyme digestion and subcloning
Subcloned into any vector and delivered as a glycerol stock and plasmid preparation

What are some examples of what directed evolution can be used for?

Increase or adjust promoter strength or specificity
Enhance or modulate protein stability
Modify or combine enzyme properties
Increase binding affinities of receptors, ligands, and antibodies
Optimize or alter signal peptide efficiencies
Destroy protein function while retaining immunogenicity
Combine and select natural polymorphisms
Increase protein half life
Adjust thermal stability

What quality criteria can I expect in GeneArt synthetic libraries?

Three criteria are important for degenerate libraries and are quality-controlled in all GeneArt degenerated libraries (except GeneArt Strings DNA Libraries):

Maximum sequence integrity of the non-degenerated parts
Maximum sequence variation of the degenerated positions with the requested nucleotide distribution
Maximum library diversity

What information do you need to provide a price estimate?

For libraries, please visit the respective home page for the library type you are interested in. On these pages you can download and fill out a specific questionnaire and send the information to geneartsupport@thermofisher.com. We will respond with a price quote and expected processing times.

What are the details of your GeneArt Strings Libraries services?

A GeneArt Strings DNA Library contains cloned linear DNA fragments with the following parameters:

200–2,000 bp in length
Up to 3 randomized regions of up to 30 bp each
The randomized regions can contain all IUPAC defined ambiguous nucleotides (N, K, S, B, …); U is not optional
Randomized regions need to be at least 30 bp from either end of the fragment and at least 30 bp from each other

GeneArt Strings DNA Libraries are not available with TRIM technology or custom nucleotide mixes. Please inquire about our other library products if you require this type of randomization.