Spatial Synthetic Cell-Free Biology

The U.S. has the biomass production potential to dramatically offset yearly petroleum consumption, but many efficiency barriers remain for developing enduring bioenergy sources. Synthetic biology allows researchers to redesign energy-relevant organisms to increase the efficiency and lower the cost of bioenergy technologies. However, developing complex gene circuit behavior in new organisms or networks can result in unexpected complications and off-target effects. Since cellular structure and scale can affect gene expression dynamics, understanding how gene expression operates within the physiological context of the cell becomes important for developing robust gene circuits. Gene expression occurs in a highly crowded and confined (from about 1 fL to several pL) environment. Macromolecules occupy 5-40% of the intracellular environment, effecting changes in molecular transport, association, and reaction rates associated with gene expression. Gene expression also exhibits “bursty” patterns of expression, characterized by episodic periods of high activity between periods of low activity. These bursting patterns are shaped not only by molecular mechanisms but also by the global availability of resources within the expression environment, both of which may be further modulated by physical effects, like crowding and confinement. Since manipulating the physical conditions surrounding gene expression can be difficult to achieve in cells, cell-free systems are used to directly probe gene expression reactions. In this work, gene expression reactions in cell-free systems are modified to mimic physiological levels of crowding and confinement, revealing information about the interplay between expression bursting, resource sharing, and spatial ordering in transcription and translation. These results explore how confined reactions alter bursting patterns and distribute limited expression resources, as well as how crowding-induced spatial inhomogeneities in transcription can affect bursting patterns in translation. The cell-free platform described here also demonstrates spatial organization of gene expression similar to that seen in cells, providing a useful technique for exploring the mechanisms of cellular self-organization in gene expression and developing spatial control over transcription and translation reactions.