ALBERT

Description: by Chelsea Virgile, Pricila Hauk, Hsuan-Chen Wu, Wu Shang, Chen-Yu Tsao, Gregory F. Payne, William E. Bentley Synthetic biologists construct innovative genetic/biological systems to treat environmental, energy, and health problems. Many systems employ rewired cells for non-native product synthesis, while a few have employed the rewired cells as ‘smart’ devices with programmable function. Building on the latter, we developed a genetic construct to control and direct bacterial motility towards hydrogen peroxide, one of the body’s immune response signaling molecules. A motivation for this work is the creation of cells that can target and autonomously treat disease, the latter signaled by hydrogen peroxide release. Bacteria naturally move towards a variety of molecular cues (e.g., nutrients) in the process of chemotaxis. In this work, we engineered bacteria to recognize and move towards hydrogen peroxide, a non-native chemoattractant and potential toxin. Our system exploits oxyRS , the native oxidative stress regulon of E . coli . We first demonstrated H 2 O 2 -mediated upregulation motility regulator, CheZ. Using transwell assays, we showed a two-fold increase in net motility towards H 2 O 2 . Then, using a 2D cell tracking system, we quantified bacterial motility descriptors including velocity, % running (of tumble/run motions), and a dynamic net directionality towards the molecular cue. In CheZ mutants, we found that increased H 2 O 2 concentration (0–200 μM) and induction time resulted in increased running speeds, ultimately reaching the native E . coli wild-type speed of ~22 μm/s with a ~45–65% ratio of running to tumbling. Finally, using a microfluidic device with stable H 2 O 2 gradients, we characterized responses and the potential for “programmed” directionality towards H 2 O 2 in quiescent fluids. Overall, the synthetic biology framework and tracking analysis in this work will provide a framework for investigating controlled motility of E . coli and other ‘smart’ probiotics for signal-directed treatment.