Directed evolution of enzymes in microfluidic devices

We are recreating the mechanisms of natural selection in the laboratory at the single enzyme level. One goal is to engineer new enzyme functions using this method. These may have interesting industrial, synthetic, or therapeutic uses. In addition, In vitro evolution also gives us the ability to observe evolutionary intermediates, control selective pressure, mutation rate, and population sizes in ways that aren’t possible using other methods, and this allows us to ask new questions about the evolutionary process itself.

Evidence for the power of evolution by natural selection is all around us in the diversity and complexity of life. Information is passed from one generation to the next in the form of genes (the genotype), and selection acts upon the products encoded by the genes (the phenotype). Populations are able to change over time due to mistakes in copying information between generations. It is these mutations that cause progeny to be slightly different than their parents, and provide the raw material for natural selection. Genes that encode phenotypes more “fit” for the environment are more likely to be passed to the next generation, and over a great number of generations, this process has led to the vast diversity of life that we see today.

We create artificial “populations”, termed libraries, of tens of millions of genes each encoding a slightly different variant of an enzyme. We are able to translate single genes into protein within emulsion drops (thus linking genotype and phenotype), and select and sort the small fraction of active variants based on their catalytic activity. The selected mutants can be mutated further and put through multiple iterations of the selection process until the desired function is achieved (see figure below).

The entire process of translation, catalysis, detection and sorting takes place in picoliter-sized emulsion drops (~15 μm diameter) formed on a microfluidic device at a rate of thousands of drops per second. This gives us the capacity to screen roughly 10⁸ unique mutants in a day using a single integrated microfluidic device.