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.

Nanometer-sized pores (nanopores) have emerged as a novel single-molecule detection technique to probe biomolecules (protein, DNA, and RNA) electrically and/or optically whilst being threaded into the pore. Since then, naturally-occurring pores known as biological nanopores (membrane proteins and bacteriophage portal proteins) along with synthetically-assembled nanopores known as solid-state nanopores have been employed to study various biomolecular characteristics. A third class of nanopores, known as hybrid nanopores, exploits the robust framework and mechanical stability of SS nanopores with atomically reproducible and tunable characteristics of biological nanopores. Biological nanopores supported by robust synthetic membrane eliminates fragility of lipid bilayers and arbitrary geometry of SS nanopores. Our group recently demonstrated the lipid insertion and stable formation of hydrophilic G20C portal protein transmembrane channels, derived from the G20C thermostable virus. Herein, we demonstrate the improved performance of our system by substituting the lipid bilayer support of the protein by thin SiN x free-standing membrane and making further modifications to chemically fix the protein on the membrane support. Finally, we report the application of our hybrid system in nanopore-based sensing of biomolecules.