Compartmentalization – A Prerequisite for Maintaining and Changing an Identity

Abstract

The chemical manipulation of DNA is much more convenient than the manipulation of the bioproducts, such as enzymes, that it encodes. The optimization of bioproducts requires cycles of diversification of DNA followed by read-out of the information into the bioproduct. Maintaining the link between the information - the genotype - and the properties of the bioproduct - the phenotype - through some form of compartmentalization is therefore an essential aspect in directed evolution. While the ideal compartment is a biological cell, many projects involving more radical changes in the bioproduct, such as the introduction of novel cofactors, may not be suitable for expression of the information in cells, and alternative in vitro methods have to be applied. Consequently, the possibility to produce simple and advanced micro compartments at high rates and to combine them with the ability to translate the information into proteins represents a unique opportunity to explore demanding enzyme engineering projects that require the evaluation of at least hundreds of thousands of enzyme variants over multiple generations.

Directed Evolution in Drops: Molecular Aspects and Applications

“…As a result, high-throughput screening techniques are necessary to achieve directed evolution, and droplet-based microfluidics now provides a way of overcoming the limiting step of screening, making it possible to achieve ∼1000-fold higher throughput than microtiter plate screening. 7 Droplets act as a picoliter-volume reaction vessel to perform biochemical assays at the single variant level, linking the activity of the functional molecule often made of amino acids (phenotype) to its corresponding encoding molecule in the form of nucleic acids (genotype). This compartmentalization is as crucial for directed evolution as it is for natural evolution since the encoding molecules and the functional molecules are distinct with the exception of DNA- and RNA-based molecules (ribozymes, DNAzymes, and aptamers).…”

“…All directed evolution experiments require a measurable activity that acts as a fitness indicator to drive the selection process “uphill” within the protein or nucleic acid sequence space, ultimately resulting in a biomolecule with a desired set of properties. As a result, high-throughput screening techniques are necessary to achieve directed evolution, and droplet-based microfluidics now provides a way of overcoming the limiting step of screening, making it possible to achieve ∼1000-fold higher throughput than microtiter plate screening . Droplets act as a picoliter-volume reaction vessel to perform biochemical assays at the single variant level, linking the activity of the functional molecule often made of amino acids (phenotype) to its corresponding encoding molecule in the form of nucleic acids (genotype).…”