Using Cell-Free Expression to Create Light-Activated Proteins In Situ in Droplet Interface Bilayer Networks

Abstract

Droplet interface bilayers (DIBs) are physical lipid bilayers that mimic real membranes in living cells, and they are formed quickly using droplets of water and lipids in oil (Fig. 1A). DIBs allow biomolecular sensing and direct detection of transmembrane proteins or peptides and small molecules such as drugs, anesthetics, or even ions. Cell-free expression systems allow in vitro protein synthesis using actual natural machinery extracted from organisms (Fig. 1B). Previous attempts to combine DIBs with cell-free extracts (CFE) encountered bilayer destabilization due to components in the expression system. This study evaluates incorporation of Promega’s T7 S30 High Yield (HY) Expression system with DIBs to pave the way for future in situ expression of light-activated bacteriorhodopsin (BR) and other complex transmembrane proteins in DIBs. A secondary output includes establishing a method for real-time monitoring and modeling of CF expression reactions using minimal volume. The ability to quantify CF output in such small volumes reduces cost per reaction from $20 to around $0.40, and synthesized protein levels reach tens to hundreds of micrograms per milliliter in less than 1 hour at 37°C.