Team:UNC-Chapel Hill

Transport of Proteins in E. coli via OMV Biogenesis

The “What” and “Why”

    Outer Membrane Vesicles (henceforth referred to as OMVs) are spherical, double-membrane enclosed structures generated naturally from a variety of both gram-negative and gram-positive bacteria, including E. coli. Typically, they serve to transport a variety of large, unwieldy molecules between cells, including proteins, lipoproteins and phospholipids. For E. coli, they measure 10-300 nm in diameter and possess the ability to fuse to the membranes of other cells, after which their contents may be imported into the cell.

    This last feature is especially of relevance, as it shows that OMVs can serve as a sort of natural transport mechanism for proteins. In turn, this allows for cell-cell transport and cell-cell communication beyond traditional methods employed in biologically-oriented sciences, including viral vectors, liposomes and quorum sensing.

    Furthermore, if such a system were to be made viable, it would have a few key advantages over other methods. Viruses would not need to be cultured and collected for effective transport between populations, with transport instead occurring naturally as a result of constant OMV production. Design issues regarding liposomes would be irrelevant, as OMVs already retain the capacity to fuse to cell membranes. Protein degradation problems typically experienced by quorum sensing, as well as programming of a receiver cell, need not apply.

    Most importantly, OMV manipulation represents a novel system that can allow any part on the registry to effectively be implemented into a fusion protein system and transported between cells within a single round of cloning. If added to the registry, it could be a boon that affects a large amount of projects in the future.

Our Research and Current Progress

    Although current research has already confirmed the ability of OMVs to incorporate select fusion proteins and transport them to other cells, no iGEM team has ever replicated this feat beyond modeling measures. Our goal, then, is to both design and test a few fusion protein systems in order to allow access to an OMV system for the registry. It can be divided up into four parts: creation of initial fusion protein constructs, assaying of protein levels in select OMVs, production of a final usable part for future registry usage and assaying of protein levels in targeted cells.

    Currently, our group has designed and cloned the majority of our fusion proteins and is set to begin OMV assays soon. In accordance with our assays, we will also work to simulate the production of OMVs and incorporation of proteins based on past and current data.