Microfluidic droplet technique for in vitro directed evolution

Increasingly over the past two decades, biotechnologists have been exploiting various molecular technologies for high-throughput screening of genes and their protein products to isolate novel functionalities with a wide range of industrial applications. One particular technology now widely used for these purposes involves directed evolution, an artificial form of evolution in which genes and proteins are evolved towards new or improved functions by imposing intense selection pressures on libraries of mutant genes generated by molecular biology techniques and expressed in heterologous systems such as Escherichia coli. Most recently, the rapid development of droplet-based microfluidics has created the potential to dramatically increase the power of directed evolution by increasing the size of the libraries and the throughput of the screening by several orders of magnitude. Here, we review the methods for generating and controlling droplets in microfluidic systems, and their applications in directed evolution. We focus on the methodologies for cell-based assays, in vitro protein expression and DNA amplification, and the prospects for using such platforms for directed evolution in next-generation biotechnologies.