Directed evolution

Directed evolution (DE, "gelenkte Evolution") is a method used in protein engineering that mimics the process of natural selection to evolve proteins or nucleic acids toward a user-defined goal. It consists of subjecting a gene to iterative rounds of mutagenesis (creating a library of variants), selection (expressing the variants and isolating members with the desired function), and amplification (generating a template for the next round). It can be performed in vivo (in living cells), or in vitro (free in solution or microdroplet). Directed evolution is used both for protein engineering as an alternative to rationally designing modified proteins, as well as studies of fundamental evolutionary principles in a controlled, laboratory environment.

Directed evolution is a mimic of the natural evolution cycle in a laboratory setting. Evolution requires three things to occur: variation between replicators, that the variation causes fitness differences upon which selection acts, and that this variation is heritable. In DE, a single gene is evolved by iterative rounds of mutagenesis, selection or screening, and amplification. Rounds of these steps are typically repeated, using the best variant from one round as the template for the next to achieve stepwise improvements.

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.

The first step in performing a cycle of directed evolution is the generation of a library of variant genes. The sequence space for random sequence is vast (10¹³⁰ possible sequences for a 100 amino acid protein) and extremely sparsely populated by functional proteins. Neither experimental, nor natural evolution can ever get close to sampling so many sequences. Of course, natural evolution samples variant sequences close to functional protein sequences and this is imitated in DE by mutagenising an already functional gene. Some calculations suggest it is entirely feasible that for all practical (i.e. functional and structural) purposes, protein sequence space has been fully explored during the course of evolution of life on Earth.

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