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

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.