Surface Display of a Unique Enzyme as a New Reporter in Synthetic Biology

Abstract

Synthetic biology is a relatively new area of engineering, with engineered gene circuits like bistable switches and oscillators being introduced about fifteen years ago. Biophysics plays a role in synthetic biology both in the analysis of the dynamics of these synthetic gene networks, as well as the synthesis of novel structural components. Here, we describe an approach to create a new structural component that also allows the investigation of synthetic gene circuit dynamics. This component allows for reporter activity such as that enabled by fluorescent proteins, while also allowing biophysical and biomaterial interactions with the cellular environment. Specifically, we have genetically engineered surface display of SNAP. The SNAP protein can bind to any benzyl guanine (BG)-conjugated molecule such as synthetic fluorescent dyes or other molecules with detectable properties (e.g., magnetic beads, which could be used in potential cell sorting approaches). SNAP is derived from O6-alkylguanine-DNA-alkyltransferase. We displayed it on the surface of E. coli cells by linking it to Lpp-OmpA, a protein created by fusing the first nine N-terminal amino acids of the lipoprotein (Lpp) to amino acids 46-159 of outer membrane protein A (OmpA), allowing fluorescent and magnetic particles to bind to the cell surface and serve as a reporter of synthetic gene network behavior. SNAP display on the surface of cells can be confirmed through microscopic observation and flow cytometry. We anticipate this new reporter will have broad impact fields ranging from cell wall and membrane biophysics to synthetic biology and material science.