Protein Directed Evolution

Creative Biolabs can assist your directed evolution projects by a rich combination of bioinformatics and DNA library synthesis.

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. Creative Biolabs synthetic libraries overcome many of the limitations of 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 CD Genomics assisted sequence design, synthetic libraries achieve thorough representation of desired variants with the specified distribution of nucleotides in areas targeted for partial degeneration or full randomization. The gene synthesis process simultaneously minimizes the introduction of unwanted mutants and erroneous changes to non-mutated portions of your constructs. This approach dramatically reduces the number of variants economizing screening time, reagents, and effort while increasing 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, statistical sequence analysis, and real-time PCR diversity analysis (control procedures are tailored to individual product lines). 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.

Creative Biolabs's Directed Evolution services cover a variety of different approaches for creating genetic variants:

Cassette mutagenesis

Cassette mutagenesis involves the cleavage by a restriction enzyme at a site in the plasmid and subsequent ligation of an oligonucleotide containing the mutation in the gene of interest to the plasmid. Usually the restriction enzyme that cuts at the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and insert to ligate to one another.

PCR site-directed mutagenesis

The same result can be accomplished using polymerase chain reaction with oligonucleotide "primers" that contain the desired mutation. As the primers are the ends of newly-synthesized strands, by engineering a mis-match during the first cycle in binding the template DNA strand, a mutation can be introduced. Because PCR employs exponential growth, after a sufficient number of cycles the mutated fragment will be amplified sufficiently to separate from the original, unmutated plasmid by a technique such as gel electrophoresis, and reinstalled in the original context using standard recombinant molecular biology techniques.

For plasmid manipulations, this technique has largely been supplanted by a PCR-like technique where a pair of complementary mutagenic primers is used to amplify the entire plasmid. This generates a nicked, circular DNA which can undergo repair by endogenous bacterial machinery. However, this process does not amplify the DNA exponentially, but linearly. Yields are complicated by the fact that the product DNA must undergo the nick repair and is not supercoiled, resulting in reduced efficiency of bacterial transformation. Finally, the product DNA is of the same size as the plasmid. Therefore, the template DNA must be eliminated by enzymatic digestion with a restriction enzyme specific for methylated DNA. The template, which for this technique should be biosynthesized will be digested, but the mutated plasmid is preserved because it was generated in vitro and is therefore unmethylated.

Directed evolution can be performed in living cells (in vitro 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.

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