(191j) Optogenetic Toolkit for Rapid and Reversible Control of Gene Expression in Bacteria

Many biological processes such as metabolism, cellular differentiation, and multicellular development involve precise coordination of gene expression. As a result, having the ability to achieve precise spatiotemporal control of gene expression is of great importance to advance our understanding on how cellular pathways function and could prove useful in biotechnological applications, including the development of cellular factories. Concomitantly, optogenetic systems offer unprecedented new ways to control gene expressions in precise spatial and temporal manner. However, current optogenetic toolbox of prokaryotes has potential issues such as lack of rapid and switchable control, less portable, low dynamic expression and limited parts. To address these shortcomings, we have recently engineered a novel bidirectional promoter system for Escherichia coli that can be induced or repressed rapidly and reversibly using a blue light dependent DNA-binding protein EL222. We demonstrated that we can positively and negatively control target genes using a single transcription factor in a single cell depending on light illumination. The light-inducible and repressible system can function in parallel with high spatial precision in a single cell and can be switched stably between ON- and OFF-states by different patterns of blue light. We have also constructed and characterized a toolbox of synthetic blue-light controllable chimeric promoters of varying strength that functions as NOT gate to precisely tune gene expression in E. coli. In addition, we have engineered two dual-mode promoters that is chemically inducible-blue light repressible. Overall, our toolbox of synthetic promoters represents a versatile and powerful platform for next-generation light-controllable synthetic biology systems in prokaryotes.