Protocol for condensate-based stabilization of gene circuit dynamics under growth-mediated dilution in E. coli

Growth-mediated dilution can destabilize synthetic gene circuits by reducing intracellular concentrations of key regulatory proteins. Here, we present a protocol that uses liquid-liquid phase separation to buffer dilution of transcription factors and stabilize synthetic gene circuits in E. coli. We describe steps for constructing phase-separating self-activation circuits, characterizing condensate material properties by fluorescence recovery after photobleaching, and imaging of condensate-promoter colocalization. We outline single-cell microscopy and population-level plate reader assays to quantify circuit activation, growth-coupled dilution dynamics, and memory retention. For complete details on the use and execution of this protocol, please refer to Zhang et al.¹.