(116c) Molecular Crowding Shaping of Gene Expression Dynamics

Synthetic biology studies have unraveled insights into gene circuit dynamics, but often neglected the impact of non-transcriptional factors, including circuit-host interactions, epigenetic factors, and intracellular microenvironments. An obvious and important non-transcriptional factor is molecular crowding, which refers to the volume exclusion effect resulting from the packing of high-density macromolecules into constrained intracellular spaces. To date, it remains unclear if and how molecular crowding can impact dynamics of gene circuits. We addressed this question by using a multi-scale synthetic biology approach by integrating single-molecule experiments, cell-free expression systems, and artificial cells. Specifically, we found that the size of crowding molecules uniquely affects both the diffusion T7 RNA polymerase and its binding to a T7 RNAP promoter. Based on the single-molecule results, we further showed that the impact of molecular crowding on gene circuits could be enhanced by weak genetic components and maximized by a negative feedback loop. Importantly, molecular crowding confers robustness of gene expression against perturbations of gene environment in both cell free systems and artificial cells. Our results have broad implications on the engineering of synthetic biological systems, gene regulation, and evolution.