GeneArt Directed Evolution FAQs

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.

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.

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 a given 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.

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.

There are a number of 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 saves time and money

• 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

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

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

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 through our online ordering system. If needed, you can then relate detailed information about your library request to our production scientists prior to starting the project.

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

Two standard options are available:

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

• 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

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

For GeneArt mutagenesis services, we will need the DNA or amino acid sequence of the gene you would like to mutagenize, the host organism you plan to use (this is important for gene optimization if you choose to include it with your request), the restriction sites you need at the 5'/3' ends and/or to avoid internally, and whether or not you want any other added motifs (e.g., Kozak sequence, stop codons, etc.) Variants can be requested online via the GeneArt portal. 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.

Variants may contain up to four of the following modifications:

• 5'/3' deletion of any length with additional 52 nt of new sequence on each side
• 5'/3' modification of maximum 52 nt on each side
• 5'/3' extension of maximum 52 nt on each side
• Internal modifications of up to 40 nt (a maximum of 3 of these internal modification blocks are allowed)
• Internal deletions of any length

Modifications (whether internal or 5'/3') have to be separated by at least 100 bp.

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.