Shaping Gene Expression in Artificial Cellular Systems by Cell-Inspired Molecular Crowding

Abstract

Synthetic biology has made tremendous recent strides in constructing artificial cellular systems using minimal cell components ex vivo, creating an experimental platform for characterizing the behavior of isolated cellular modules and a form of biotechnology for the controlled operation of artificial cells. The robustness and efficiency of these systems are nonetheless challenging to control, in part because artificial cells establish an environment that is still very different from that of actual living cells. Here, we present a novel approach towards bridging the gap between artificial and true cell environments by developing artificial cells incorporating controlled macromolecular crowding, mimicking a key feature of natural cells known to dramatically influence biochemical kinetics. We demonstrate the value of our approach by showing that molecular crowding enhances gene expression and confers robustness against perturbations of gene environments. We further elucidate the underlying mechanisms of these phenomena at the single molecule level by demonstrating how large crowding molecules decrease diffusion of T7 RNA polymerase, but increase its binding to a T7 RNAP promoter. Based on single-molecule results, we further show that the impact of molecular crowding on gene circuits is enhanced by weak genetic components and maximized by a negative feedback loop. By bridging a key gap between artificial cell technology and the environment of living cells, we demonstrate the importance of intracellular crowding to efficient and robust function of biological circuits and suggest new engineering principles for controlled modulation of synthetic genetic systems.