Automated Synthetic Cell-based Screening for Designed Proteins with Emergent Functions

Abstract

Designing minimal biological systems with emergent functions such as spatiotemporal self-organization is a central goal of bottom-up synthetic biology. While computational optimization and design show promises to accelerate functional protein engineering through Design-Build-Test-Learn cycles, screening libraries for complex functions remains a major challenge. Conventional screens typically lack the spatiotemporal resolution and cell-like confinement required in bottom-up synthetic biology. Here, we present PUREdrop, an automated microfluidic platform that encapsulates and expresses protein libraries in thousands of picolitre-sized synthetic cells per construct. These droplets are sorted into a 96-well plate and analyzed by time-lapse imaging, allowing parallel quantification of expression kinetics and emergent functions. To demonstrate the platforms potential, we first screened computationally re-designed variants of the bacterial cell division protein FtsZ, identifying variants with improved bundling phenotypes and faster kinetics. We then extended our screening procedure towards general protein modulators of FtsZ and identified a combination that anchors filaments to the interface, producing a ring-like phenotype. PUREdrop bridges computational protein engineering and synthetic cell research, elevating the rational engineering of complex biological function to the next level.