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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. 

Abstract The field of synthetic biology and biosystems engineering increasingly acknowledges the need for a holistic design approach that incorporates circuit-host interactions into the design process. Engineered circuits are not isolated entities but inherently entwined with the dynamic host environment. One such circuit-host interaction, ‘growth feedback’, results when modifications in host growth patterns influence the operation of gene circuits. The growth-mediated effects can range from growth-dependent elevation in protein/mRNA dilution rate to changes in resource reallocation within the cell, which can lead to complete functional collapse in complex circuits. To achieve robust circuit performance, synthetic biologists employ a variety of control mechanisms to stabilize and insulate circuit behavior against growth changes. Here we propose a simple strategy by incorporating one repressive edge in a growth-sensitive bistable circuit. Through both simulation and in vitro experimentation, we demonstrate how this additional repressive node stabilizes protein levels and increases the robustness of a bistable circuit in response to growth feedback. We propose the incorporation of repressive links in gene circuits as a control strategy for desensitizing gene circuits against growth fluctuations.