(75e) A Library of Inducible Synthetic Devices for Phenotype Regulation In Prokaryotes

Implementing an original workflow, we have designed, constructed and characterized a library of new synthetic molecular devices to achieve an assortment of inducible gene expression phenotypes in prokaryotes. We initially focused our efforts on two simple synthetic transcription activators, prokaryotic-TetOn and prokaryotic-TetOff. These synthetic devices switch gene expression on in response to the presence or the absence of anhydrotetracycline (respectively). Using these devices, in combination with naturally occurring regulatory components, we subsequently designed, built and tested other synthetic devices to achieve a range of functionalities.

For each desired phenotype, we first selected the appropriate device type and the protein and DNA components to use. Next, we optimized the geometries of these components and the overall device using molecular modeling. Through this, we achieved an unprecedented combination of controllable and transactivating functions with each system. We then built the devices and tested their behavior using flow cytometry. Last, we implemented stochastic simulations to quantify the strength of the key biomolecular interactions arising in each system.

The unique functions of our systems were found to be robustly tight and covering a wide range of dynamic gene expression phenotypes. When induced, they activate gene expression many-fold relative to their uninduced states. The new devices presented here may become useful additions to the molecular toolboxes used by biologists to control bacterial gene expression. Our methodology may also be a foundation for the design, development and characterization of additional synthetic device libraries.