Novel bi-lamellar lipid vesicles for studying double-membrane transenvelope proteins

ended

Double membranes are ubiquitous throughout the domains of life, accommodating remarkable protein machineries which are fundamental to the cellular activity. However, the study of these proteins is restricted by the lack of a suitable membrane model to accommodate them. Within the framework of BiLamVesicles I will develop a novel bi-lamellar lipid vesicle as a tool for hosting and studying proteins which naturally span across double membranes such as the nucleus and Gram-negative bacteria envelopes. To integrate the protein of choice within the vesicle envelope I will design and employ a highly regulated layer-by-layer assembly in a microfluidic chip. This approach will combine the host’s expertise in microfluidics and biophysics with my expertise in surface interactions and surface chemistry to allow an exquisite control over the membrane composition of bi-lamellar vesicles and the protein insertion process. Once assembled, I will use these vesicles to study the activity of the entire Gram-negative bacterial transporter system AcrAB-TolC, an archetype multidrug efflux pump of Escherichia coli. I will spatially isolate vesicles in a microfluidic chip and directly quantify transport rates through a full efflux pump system at the single-vesicle-level for the first time, using an advanced optofluidic system. The synergy between microfluidics and the proposed double membrane vesicles will produce a ground-breaking biotechnological technique for studying the activity of as yet inaccessible proteins in a biologically-relevant environment. This research will stretch the existing boundaries set by current membrane models and will pave the way for developing advanced techniques for drug screening assays.