A New Embedded Process for Compartmentalized Cell‐Free Protein Expression and On‐line Detection in Microfluidic Devices

During the last century, an enormous number of protein functions were identified, many of which can be generally described as catalytic. Ongoing research currently focuses on two aspects, analysis of the native proteome to reveal new cellular functions, and adaptation of proteins with known functions to technical processes, with the additional aspect of how these functions could be modified or improved. To this end, directed evolution of proteins has in many cases proved to be a successful strategy for designing new biocatalysts for chemical, pharmaceutical or even household use. It relies on the sensitive detection of mutants with new or improved properties, as well as their efficient singling out and amplification. In this work, a combined approach based on ultrasensitive spectroscopy, microfluidic chips and artificial cells is provided, in order to contribute a new analytical tool for both proteome discovery and evolutionary biotechnology. What is the artificial cell concept? Artificial cells reduce the features of a complex microbiological organism down to the two basic properties that are needed for defined protein analysis: compartmentalization and in vitro protein expression. A promising approach for compartmentalization in miniature environments is the formation of a water-in-oil emulsion, in which micrometer-sized water droplets embedded in a hydrophobic layer serve as artificial biocontainers. All essential compounds for transcription and translation, including the protein-encoding gene as well as a substrate to probe for catalysis, can be included within the same compartment, thus providing an essential requirement for in vitro evolution by linking genotype and phenotype. However, with classical methods for the generation of emulsions in batch (e.g. magnetic stir bar), individual droplets cannot be accurately addressed, and automated droplet handling, analysis and sorting is limited. For in vitro protein evolution, tools for automated highthroughput formation, analysis and isolation of individual biocontainers are highly desirable. 10] Therefore downscaling and integration of all functional steps on a single microchip represents an exciting advance. In microfluidic channel networks, the formation of water-in-oil emulsions at high rates and with monodisperse droplets becomes feasible, while small sample quantities can be controllably introduced, while automated droplet handling, sensitive detection of products and potential isolation of individual droplets can be performed. In this work, we demonstrate the efficient implementation of microstructured devices to generate water-in-oil emulsions, and at the same time perform in vitro expression of proteins inside the water droplets (Figure 1). By using the red-shifted