Self-organization controls expression more than abundance of molecular components of transcription and translation in confined cell-free gene expression

Cell-free gene expression using purified components or cell extracts has become an important platform for synthetic biology that is finding a growing number of practical applications. Unfortunately, at cell-relevant reactor volumes, cell-free expression suffers from excessive variability (noise) such that protein concentrations may vary by more than an order of magnitude across a population of identically constructed reaction chambers. Consensus opinion holds that variability in expression is due to the stochastic distribution of expression resources (DNA, RNAP, ribosomes, etc.) across the population of reaction chambers. In contrast, here we find that chamber-to-chamber variation in the expression efficiency generates the large variability in protein production. Through analysis and modeling, we show that chambers self-organize into expression centers that control expression efficiency. Chambers that organize into many centers, each having relatively few expression resources, exhibit high expression efficiency. Conversely, chambers that organize into just a few centers where each center has an abundance of resources, exhibit low expression efficiency. A particularly surprising finding is that diluting expression resources reduces the chamber-to-chamber variation in protein production. Chambers with dilute pools of expression resources exhibit higher expression efficiency and lower expression noise than those with more concentrated expression resources. In addition to demonstrating the means to tune expression noise, these results demonstrate that in cell-free systems, self-organization may exert even more influence over expression than the abundance of the molecular components of transcription and translation. These observations in cell-free platform may elucidate how self-organized, membrane-less structures emerge and function in cells.