Spatial engineering ofE. coliwith addressable phase-separated RNAs

Abstract Biochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here we demonstrate Transcriptionally Engineered Addressable RNA Solvent droplets (TEARS) as synthetic microdomains within the Escherichia coli. TEARS are assembled from RNA-binding protein recruitment domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures and a non-equilibrium mechanism controlling their composition and reaction robustness. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates and scaffolding protein-protein interactions. TEARS are a simple and versatile tool for spatially controlling E. coli biochemistry.