Directed evolution of biofilm production using double emulsion droplets

Directed evolution offers a powerful approach to enhance biological phenotypes through iterative randomization and selection, traditionally applied to proteins but increasingly relevant to whole organisms. In this study, we introduce a novel method integrating microfluidic-generated double emulsion (DE) droplets with flow cytometry and fluorescence-activated cell sorting to enable high-throughput directed evolution under precisely controlled conditions. Using Acinetobacter baumannii (A. baumannii) as a biological example—known as a biofilm producer, we encapsulated single cells in DE droplets and sorted them based on a fluorescence marker tied to the biofilm-forming phenotype. The sorted cells were re-cultured in bulk, re-encapsulated, and re-analyzed by flow cytometry. This approach successfully enriched A. baumannii variants, and the selected phenotypes persisted after bulk regrowth. Unlike traditional methods, which are limited by low throughput or shear-induced damage, our DE droplet system achieves quantitative analysis at rates up to a million events per hour while preserving cell integrity. To our knowledge, this is the first application of DE droplets with flow cytometry for directed evolution on whole organisms. We believe this method opens new avenues for linking phenotypes to genotypes across multiple rounds of evolution, advancing studies of complex microbial traits.