The assembly of channel proteins into vesicle membranes is a useful strategy to control activities of vesicle-based systems. Here, we developed a membrane AND gate that responds to both a fatty acid and a pore-forming channel protein to induce the release of encapsulated cargo. We explored how membrane composition affects the functional assembly of α-hemolysin into phospholipid vesicles as a function of oleic acid content and α-hemolysin concentration. We then showed that we could induce α-hemolysin assembly when we added oleic acid micelles to a specific composition of phospholipid vesicles. Finally, we demonstrated that our membrane AND gate could be coupled to a gene expression system. Our study provides a new method to control the temporal dynamics of vesicle permeability by controlling when the functional assembly of a channel protein into synthetic vesicles occurs. Furthermore, a membrane AND gate that utilizes membrane-associating biomolecules introduces a new way to implement Boolean logic that should complement genetic logic circuits and ultimately enhance the capabilities of artificial cellular systems.

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

Targeted vesicle fusion is a promising approach to selectively control interactions between vesicle compartments and would enable the initiation of biological reactions in complex aqueous environments. Here, we explore how two features of vesicle membranes, DNA tethers and phase‐segregated membranes, promote fusion between specific vesicle populations. Membrane phase‐segregation provides an energetic driver for membrane fusion that increases the efficiency of DNA‐mediated fusion events. The orthogonality provided by DNA tethers allows us to direct fusion and delivery of DNA cargo to specific vesicle populations. Vesicle fusion between DNA‐tethered vesicles can be used to initiate in vitro protein expression to produce model soluble and membrane proteins. Engineering orthogonal fusion events between DNA‐tethered vesicles provides a new strategy to control the spatiotemporal dynamics of cell‐free reactions, expanding opportunities to engineer artificial cellular systems.

Polyunsaturated fatty acids (PUFAs) modify the activity of a wide range of membrane proteins and are increasingly hypothesized to modulate protein activity by indirectly altering membrane physical properties. Among the various physical properties affected by PUFAs, the membrane area expansion modulus (K a ), which measures membrane strain in response to applied force, is expected to be a significant controller of channel activity. Yet, the impact of PUFAs on membrane K a has not been measured previously. Through a series of micropipette aspiration studies, we measured the apparent K a (K app ) of phospholipid model membranes containing nonesterified fatty acids. First, we measured membrane K app as a function of the location of the unsaturated bonds and degree of unsaturation in the incorporated fatty acids and found that K app generally decreases in the presence of fatty acids with three or more unsaturated bonds. Next, we assessed how select u-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), affect the K app of membranes containing cholesterol. In vesicles prepared with high amounts of cholesterol, which should increase the propensity of the membrane to phase segregate, we found that inclusion of DHA decreases the K app in comparison to EPA. We also measured how these u-3 PUFAs affect membrane fluidity and bending rigidity to determine how membrane K app changes in relation to these other physical properties. Our study shows that PUFAs generally decrease the K app of membranes and that EPA and DHA have differential effects on K app when membranes contain higher levels of cholesterol. Our results suggest membrane phase behavior and the distribution of membrane-elasticizing amphiphiles impact the ability of a membrane to stretch.

While current research is centered on observing biophysical properties and phenomena in giant unilamellar vesicles (GUVs), little is known about fabrication parameters that control GUV formation. Using different lipids and rehydration buffers, we directly observe varying dynamics of hydrogel-assisted GUV formation via fluorescence microscopy. We observe the effects of buffer ionic strength, osmolarity, agarose density, and pH on the formation of GUVs using neutral and charged lipids. We find that increasing rehydration buffer ionic strength correlates with increased vesicle size and rate of GUV formation. Increasing buffer acidity increased the rate of GUV formation, while more basic environments slowed the rate. For buffers containing 500 mM sucrose, GUV formation was overall inhibited and only tubules formed. Observations of GUV formation dynamics elucidate parametric effects of charge, ionic strength, pH, and osmolarity, demonstrating the versatility of this biomimetic platform.