S147: Computer-aided design of synthetic biological protein devices

Implementing 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 (proTeOn) and prokaryotic-TetOff (proTeOff), were built by fusing variants of the tetracycline repressor, TetR, with the transactivating domain of the LuxR activator. A DNA promoter sequence complementary to these protein domains was also designed. It includes TetR’s operator, tetO, and elements of the lux promoter. First, the geometry of the molecular components was optimized using molecular modeling to best achieve an unprecedented combination of controllable and transactivating functions 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 the expression of bacterial genes. 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.