Giant Vesicles Mimicking Outer Membrane of Gram-Negative Bacterium

Abstract

Engineering of synthetic cells which mimics the native membrane opens up various platforms for research in membrane biology as well as synthetic biology field. In this work, giant unilamellar vesicles (GUV) mimicking outer membrane of Gram-negative bacterium is engineered by incorporating lipopolysaccharide (LPS) under physiological salt condition. We successfully incorporated LPS in Gram-negative (GN) vesicles by inverse-phase precursor film assembly using gel-assisted technology and confirmed at single-vesicle level using fluorescence detection system. Effective integration of LPS in vesicles is attained in presence of Ca2+ ions at a ratio of LPS: E. coli polar lipid extract (GN extract) (1:6) to construct outer membrane mimicking (OMM) vesicles comparable to the bacterial outer-membrane composition. Quantitative transport rates of antibiotic showed a much slower flux across vesicles with LPS (OMM) compared to GN vesicles further confirming the incorporation of LPS in vesicles and its significance in native bacterial cells. To demonstrate the functionality of engineered OMM vesicles, a model peptide melittin is reconstituted in the membrane of both vesicles and the uptake of small different sized molecules is studied. In GN vesicles, a steady uptake of large molecules is observed in peptide: lipid (1:15) ratio, where in OMM vesicles pore formation and diffusion of molecules occurred at higher peptide: lipid (1:5) ratio confirming intrinsic resistance of OMM vesicles due to presence of LPS. The OMM vesicles constructed here will find immense applications in fundamental understanding of membrane biophysical phenomena.