Design a Tunable Expression and Display System on Bacterial Spore Surface

Bacterial spores have been explored to display proteins of interest as it offers potential advantages compared to soluble systems or other displaying platforms. First, spores are robust structures that are able to survive extreme temperatures, desiccation, and exposure to solvents and noxious chemicals. A few proteins displayed on the surface of Bacillus spores have shown enhanced stability. Second, the displayed proteins are part of the spore and they are synthesized and assembled during the sporulation process. Therefore, no protein purification is needed other than collecting the spores, which will significantly reduce the production costs of proteins. Third, enzymes displayed on the spore surface are reusable and recyclable. Spore serves as an immobilization platform and allows for multiple rounds of reactions. In addition, these spores are viable and able to germinate into cells. So spores with decreased enzyme activity can be easily regenerated through a germination-sporulation cycle. We have been working on displaying multiple proteins on the spore surface to create miniature reactors with complex functions. One challenge is that the expression level of the spore surface proteins is tightly regulated and no inducible expression system is available in response to external signals. In this work, we designed a few hybrid promoters by incorporating operators for commonly used repressors into the promoters for a few spore surface proteins. The resulted promoters have the natural regulatory elements allowing for the timely expression during the sporulation process and also the operators controlling the expression level through the level of the inducers. The designed tunable expression and display system will greatly facilitate the study and engineering of multiply protein complexes on the spore surface.