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Directed Evolution
Droplet microfluidics has been used for directed evolution. Directed evolution is a method used to develop novel proteins, pathways and genomes through a series repetitions of library generation and subsequent screening to obtain a desired phenotype; this allows scientists to engineer proteins without advanced knowledge of the structure and functions of proteins (i.e., rational design). Due to the iterative nature of directed evolution and the necessity for large libraries, directed evolution at the macroscale can be a costly endeavor. As such, performing experiments at the microscale through droplet-based microfluidics provides a significantly cheaper alternative to macroscopic equivalents. Various approaches price the directed evolution through droplet microfluidics under $40 for a screen of a 106-107 sized gene library, while the corresponding macroscale experiment is priced at approximately $15 million. Additionally, with screening times that range from 300 to 2000 droplets sorted per second, droplet-based microfluidics provides a platform for significantly accelerated library screening such that gene libraries of 107 can be sorted well within a day. Droplet-based microfluidic devices make directed evolution accessible and cost effective.

Many different approaches to device construction of droplet-based microfluidic devices have been developed for directed evolution in order to have the capacity to screen a vast variety of different proteins, pathways, and genomes. One method of feeding libraries into the microfluidic device uses single cell encapsulation, in which droplets contain a maximum of one cell each. This avoids confounding results that could be generated by having multiple cells, and consequently multiple genotypes, in a single droplet, while maximizing the efficiency of resource consumption. This method enables the detection of secreted proteins and proteins on the cell membrane. The addition of a cell lysate to the droplets, which breaks down the cellular membrane such that the intracellular species are freely available within the droplet, expands the capabilities of the single cell encapsulation method to analyze intracellular proteins. The library can also be made entirely in vitro (i.e., not in its biological/cellular context) such that the content of the droplet is exclusively a mutated DNA strand. The in vitro system requires PCR and the use of in vitro transcription and translation (IVTT) systems to generate the desired protein in the droplet for analysis. Sorting of droplets for directed evolution is primarily done by fluorescence detection (e.g., fluorescence-activated droplet sorting (FADS)), however recent developments in a absorbance-based sorting methods, known as absorbance-activated droplet sorting (AADS), have expanded the diversity of substrates that can undergo directed evolution through a droplet-based microfluidic device. Recently, sorting capability has even expanded to the detection of NADPH levels and has been used to create higher activity NADP-dependent oxidoreductases. Ultimately, the potential for different methods of droplet creation and analysis in directed evolution droplet-based microfluidic devices allows for a variability that facilitates a large population of potential candidates for directed evolution.

As a method for protein engineering, directed evolution has many applications in fields from development of drugs and vaccines to the synthesis of food and chemicals. A microfluidic device was developed to identify improved enzyme production hosts (i.e., cell factories) that can be employed industrially in various fields. An artificial aldolase was further enhanced by 30-fold using droplet-based microfluidics so that it’s activity resembled that of naturally occurring proteins. More recently, the creation of functional oxidases has been enabled by a novel microfluidic device created by Debon et al. The droplet-based microfluidic approach to the directed evolution has a great potential for the development of a myriad of novel proteins.