A membrane logic gate to control secretion from vesicles

Statement of Purpose: There has been considerable interest in the design of synthetic vesicles that can both synthesize and deliver bioactive payloads for technological and therapeutic applications. Channel proteins, such as α-hemolysin (αHL), that integrate into vesicle membranes provide a strategy to release encapsulated molecules. In addition, channel proteins can be expressed via cell-free expression systems such that channel functions can be coupled to genetic circuits. It has been difficult, however, to control precisely when channel proteins integrate into vesicle membranes. Here, we present the design of a membrane AND gate in which the presence of both a membrane-associating lipid and a channel protein lead to the functional assembly of the channel protein into phospholipid vesicle membranes. Using dye release assays, we characterized the propensity for αHL to integrate into vesicles as a function of cholesterol and OA content, and αHL concentration. We explored how the addition of OA micelles to phospholipid vesicles enables changes in membrane composition that promote pore formation. Finally, we explored the compatibility of our membrane design with a cell-free expression system in which αHL is expressed.