Refactoring functional Type III Secretion system in Salmonella

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Mi Ryoung Song, David Sukovich, Ethan Mirsky, and Christopher Voigt

Massachusetts Institute of Technology, United States

The type III secretion system (TTSS) is a needle-shaped export machine, which directly delivers proteins from bacterial cytosol to host cytoplasm. The expressions of genes encoding those proteins are tightly regulated temporarily and spatially under physiological conditions. Here, we refactored two major gene clusters, prg-org or inv-spa clusters, encoding the structural components of TTSS to eliminate all native regulation for the expression of system in Salmonella. During the refactoring procedures, non-coding DNAs, non-essential genes, and transcription factors were removed then the codons of essential genes were randomized. These were organized into operons under the control of synthetic ribosomal binding site (RBS) for each gene. For the control of expression of refactored operons, we used inducible orthogonal bacteriophage RNA polymerase systems. Using these regulated refactored operons, we could detect secreted effectors from bacterial culture media as an indicative hallmark of functional TTSS. This work allows us to take advantage of most effective protein export system without limitation by native regulatory complexities through the refactoring procedure for diverse applications in biotechnology.