proTeOn and proTeOff, New Protein Devices That Inducibly Activate Bacterial Gene Expression

Using an original workflow, we have modeled, constructed, and characterized two new molecular devices that inducibly activate gene expression in Escherichia coli. The devices, prokaryotic-TetOn and prokaryotic-TetOff, were built by fusing an inducible DNA-binding protein domain to a transcription activation domain and constructing a complementary synthetic promoter sequence through which they could control downstream gene expression. In particular, the transactivators were built using variants of the tetracycline repressor, TetR, and the transactivating domain of the LuxR activator. The complementary promoter sequence included TetR's operator, tetO, and elements of the lux promoter. These specific protein domains and their operator sites were chosen as they have been thoroughly studied and well characterized. First, our methodology began with optimizing the geometry of the molecular components using molecular modeling. We did so to achieve an unprecedented combination of controllable and transactivating function in bacterial organisms. The devices were then built to activate the expression of green fluorescent protein. Their unique function was found to be robustly tight and activating many-fold increases of expressed gene levels, as measured by flow cytometry experiments. The devices were further characterized with stochastic kinetic models. The new devices presented herein may become useful additions to the molecular toolboxes used by biologists to control bacterial gene expression. The methodology used may also be a foundation for the design, development, and characterization of a library of such devices and more complex gene regulatory networks.