Phase separation to buffer growth-mediated dilution in synthetic circuits.

Abstract

Fluctuations in host cell growth pose a critical challenge for maintaining reliable function in synthetic gene circuits. Growth-mediated dilution causes a global reduction in circuit component concentrations, which can significantly destabilize circuit behavior. However, effective strategies to counteract this problem remain lacking. Here, we present a phase-separation-based strategy to directly mitigate dilution effects. By fusing transcription factors (TFs) to intrinsically disordered regions (IDRs), we drive the formation of transcriptional condensates that concentrate TFs at their target promoters. These condensates buffer against prolonged rapid dilution of TF concentration and preserve bistable memory in self-activation circuits across variable growth conditions. We further show that this approach improves production efficiency in a cinnamic acid biosynthesis pathway. Together, our results establish liquid-liquid phase separation as an emerging design principle for constructing resilient synthetic circuits that maintain robust performance under dynamic growth conditions.