A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution

Abstract

In vitro screening systems based on the coupled transcription and translation of genes using cell-free systems have a number of attractive features for protein engineering and directed evolution. We present a completely in vitro ultrahigh-throughput screening platform using droplet-based microfluidics. Single genes are compartmentalized in aqueous droplets, dispersed in inert carrier oil, and amplified using the polymerase chain reaction (PCR). After amplification, the droplets, now containing 30,000 copies of each gene, are fused one-to-one with droplets containing a cell-free coupled transcription-translation (IVTT) system and the reagents for a fluorogenic assay. Fluorescence-activated electrocoalescence with an aqueous stream is then used to selectively recover genes from droplets containing the desired activity. We demonstrate, by selecting mixtures of lacZ genes encoding the enzyme β-galactosidase and lacZmut genes encoding an inactive variant, that this system can sort at 2000 droplets s(-1): lacZ genes were enriched 502-fold from a 1 : 100 molar ratio of lacZ : lacZmut genes. Indeed, the false positive and false negative error rates were both <0.004 and the results indicate that enrichment is not limited by the sorting efficiency, but by the co-encapsulation of multiple genes in droplets, which is described by the Poisson distribution. Compared to screening using microtiter plate-based systems, the volume and cost of PCR and IVTT reagents are reduced by almost 10(5)-fold, allowing the screening of 10(6) genes using only 150 μL of reagents.

Dissecting enzyme function with microfluidic-based deep mutational scanning

“…Droplet-based microfluidic screening provides a flexible platform for assaying enzyme activity over a broad range of reaction conditions (10)(11)(12)(13). We adapted our screening protocol to include a heat challenge and enriched for mutations that increase the enzyme's thermostability.…”

“…Through comprehensive mutagenesis and functional characterization of this enzyme, we were able, with minimal bias, to discover residues crucial to function and identify mutations that enhance its activity at elevated temperatures. This approach can be applied to any enzyme whose chemical activity can be measured with a fluorogenic assay in microfluidic droplets (10)(11)(12)(13). Our method extends the applicability of deep mutational scanning to a wide range of protein functions and reaction conditions not accessible by other high-throughput methods.…”

Enhanced transcription rates in membrane-free protocells formed by coacervation of cell lysate

“…Among the most important complex sets of reactions in the cell is protein synthesis (the result of transcription and translation), a process that functions in vitro despite almost two orders of magnitude lower total protein concentration than found in vivo. We and others have previously shown how droplet-based microfluidic devices can be used to study the kinetics of in vitro expression of a reporter protein using commercial in vitro transcription and translation kits (21)(22)(23). The volume of droplets can be controlled via osmotic transport of water to and from reservoir channels filled with concentrated salt solutions (24,25) and separated from the droplet traps (25) by a thin (15-μm) polydimethylsiloxane (PDMS) membrane (Fig.…”

PCR digitale en micro-compartiments

“…De plus, l'analyse de centaines d'échantillons en parallèle permet de rentabiliser le prix de chaque puce. Le champ de la microfluidique promet de nombreux avantages tels que l'automatisation de processus complexes successifs [39]. La commercialisation…”