The chemical transformations of biology occur not in the test tube, but compartmentalized inside the cell membrane. Systematically studying this compartmentalized chemistry and harnessing its benefits for therapeutic applications through directed enzyme evolution will require methods for controlled synthesis and functional screening of cell-like compartments. Mentored research activity will significantly expand on current efforts in microfluidic directed evolution systems by exploring circuitry for the controlled high-throughput synthesis of monodisperse emulsions and lipid vesicles for in vitro compartmentalization (IVC). Vesicles will compartmentalize individual molecules from a population of ligase ribozymes; compartmentalization will permit a thorough exploration of the fitness landscape by prohibiting a single advantageous genotype from dominating the reaction and will permit selection for trans-acting, multiple-turnover ribozymes. This will serve as the foundation for an independent research program. The microfluidic IVC (?lVC) processor throughput will be increased to 1x109 per hour by arraying vesicle generation channels. For experiments requiring droplet screening, confocal fluorescence scanning of 2D vesicle arrays and sorting of the array will be accomplished either by multiplexed membrane valve arrays or dielectrophoresis using electrode arrays; ?-galactosidase and a-hemolysin will serve as models for using the ?lVC processor to evolve cytosolic and transmembrane protein targets. Long-term research program goals for the ?lVC processor include evolving membrane receptors (e.g., CCR5 and CD4) in liposomes, selecting for enhanced binding of envelope protein-receptor complexes, evolving membrane-bound ligands for liposomes used in the targeted delivery of therapeutics, and evolving transporters for selective encapsulation of neurotoxins. ? ? ?
| Matosevic, Sandro; Paegel, Brian M (2013) Layer-by-layer cell membrane assembly. Nat Chem 5:958-63 |
