Controlled transport of molecules across boundaries for energy exchange, sensing, and communication is an essential step toward cell-like synthetic systems. This communication between the gene expression compartment and the external environment requires reaction chambers that are permeable to molecular species that influence expression. In lipid vesicle reaction chambers, species that support expression -from small ions to amino acids -may diffuse across membranes and amplify protein production. However, vesicle-to-vesicle variation in membrane permeability may lead to low total expression and high variability in this expression. We demonstrate a simple optical treatment method that greatly reduces the variability in membrane permeability. When transport across the membrane was essential for expression, this optical treatment increased mean expression level by ~6-fold and reduced expression variability by nearly two orders of magnitude. These results demonstrate membrane engineering may enable essential steps toward cell-like synthetic systems. The experimental platform described here provides a means of understanding controlled transport motifs in individual cells and groups of cells working cooperatively through cell-to-cell molecular signaling.
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.
Essential steps toward synthetic cell-like systems require controlled transport of molecular species across the boundary between encapsulated expression and the external environment. When molecular species (e.g. small ions, amino acids) required for expression (i.e. expression resources) may cross this boundary, this transport process plays an important role in gene expression dynamics and expression variability. Here we show how the location (encapsulated or external) of the expression resources controls the level and the dynamics of cell-free protein expression confined in permeable lipid vesicles. Regardless of the concentration of encapsulated resources, external resources were essential for protein production. Compared to resource poor external environments, plentiful external resources increased expression by ~7fold, and rescued expression when internal resources were lacking. Intriguingly, the location of resources and the membrane transport properties dictated expression dynamics in a manner well predicted by a simple transport-expression model. These results suggest membrane engineering as a means for spatio-temporal control of gene expression in cell-free synthetic biology applications and demonstrate a flexible experimental platform to understand the interplay between membrane transport and expression in cellular systems.
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