Protein production is a growing, multi-billion dollar industry that supplies tailored enzymes, such as those used in pharmaceuticals, detergents, textile manufacturing, and environmentally-friendly chemical production. New methods, utilizing bacteria as "protein factories", have the potential to dramatically reduce manufacturing costs and the development time for new protein products. A critical barrier to realizing these benefits is identification of a cost-effective way to separate desired functional proteins from the remaining contents of their host cells. Secreting protein products directly out of the cell is a promising method toward this goal. This work proposes to engineer a protein secretion system in a laboratory strain of bacteria, Salmonella enterica, for efficient and low-cost production of proteins. Success would produce a path for agile design and production of proteins for a number of industries. Moreover, this work will train students at all levels for careers in biotechnology, as the investigators will participate in programs bringing cutting-edge science related to this work to the general public via museums, community talks, and science festivals. <br/><br/>Controlled transport of proteins from the inside of the cell to the outside is a challenge that nature has overcome in several ways, but that scientists have yet to fully explore. This proposal focuses on engineering the type III secretion system of Salmonella enterica to secrete non-native proteins at high quantity and low cost. The specific objectives are to 1) increase the protein yields achieved with bacterial production and secretion, 2) optimize control over the secretion process to enable scale up to industrial scales, and 3) develop a suite of molecular biology tools for the high-throughput production of a broad set of proteins. Transforming the type III secretion system to enable efficient production of desired proteins will improve understanding of the mechanisms governing this process. Such information is critical not only to molecular biologists as they construct a detailed picture of cellular function, but also to synthetic biologists who wish to engineer organisms with greater levels of complexity and extracellular interaction than previously possible. This work will result in new constructs, protocols, and strains to be shared with other scientists for protein production at both the industrial and research scales.