Engineering high-precision, dynamic genetic control systems via supercoiling-guided design

Integrating synthetic circuitry into larger transcriptional networks to mediate predictable cellular behaviors remains a challenge within synthetic biology. Rational de novo design of synthetic circuits for cell engineering remains challenging. In particular, the stochastic nature of transcription makes coordinating expression across multiple genetic elements challenging. To address this challenge, my lab recently developed a theoretical framework for exploring how DNA supercoils—dynamic structures induced during transcription—influence transcription and gene expression in synthetic and native gene systems. We find that gene syntax—the relative ordering and orientation of genes—defines the expression profiles, variance, burst dynamics, and intergene correlation of two-gene systems. By applying our model to both a synthetic toggle switch and the endogenous zebrafish segmentation network, we find that supercoiling can enhance or weaken conventional biochemical regulatory strategies such as mRNA- and protein-mediated feedback loops. In cell culture, we confirmed that two-gene circuits qualitatively match the syntax-specific profiles predicted by our model. Our model integrates supercoiling-mediated biophysical feedback with classic gene regulation motifs such as transcriptional repressors that are well-studied in native and synthetic contexts. Our model provides an extensible framework for modeling an arbitrary number of genes and regulatory architectures. Our results suggest that supercoiling couples behavior between neighboring genes, representing a novel regulatory mechanism. Additionally, our predictions suggest why some circuit designs fail and provide a path to improving transgenic designs. Harnessing the insights from our model will enable enhanced transcriptional control, providing a robust method to tune expression levels, dynamics, and noise needed for the construction of transgene systems including synthetic gene circuits in primary cells and diverse cell engineering applications including cellular reprogramming.