Abstract
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 H2O2-mediated upregulation motility regulator, CheZ. Using transwell assays, we showed a two-fold increase in net motility towards H2O2. 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 H2O2 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 H2O2 gradients, we characterized responses and the potential for “programmed” directionality towards H2O2 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.
Highlights
Bacteria naturally respond to oxidative stressors such as hydrogen peroxide and other reactive oxygen species (ROS) that are released by eukaryotic cells upon insult such as pathogen
In order to attract our engineered bacteria toward to a localized injury marked by the presence of hydrogen peroxide, we developed a system that guides E. coli swimming towards H2O2 by controlling the (i) ratio of run to tumble, and (ii) cell velocity in the presence of an H2O2 gradient
Using the same null mutants transformed with the oxyRS induced CheZ vector, pZY1-oxyR-poxyS-cheZ, (HCW01-pHW02) an increasing ring size was observed with increasing H2O2 from 0 to 300 μM (2.5–6.5 cm)
Summary
Bacteria naturally respond to oxidative stressors such as hydrogen peroxide and other reactive oxygen species (ROS) that are released by eukaryotic cells upon insult such as pathogen. Hydrogen peroxide-guided bacterial pseudotaxis tolerate, rapidly consume, and swim in response to increased H2O2 without suffering significant oxidative stress Such engineered H2O2-controlled bacteria may allow for a wide range of application for future therapies or applications. Over time, when cells swim in the presence of a CheZ-inducing molecular stimulant and do not swim in the absence of the same stimulant, there will be a net propensity for the persistence of cells in the presence of the stimulant This is a process of pseudotaxis [21, 33,34,35] as directionality can be “programmed” for a signal molecule not recognized by chemotaxis receptors and regulators, but by the controlled generation of a concentration gradient. While demonstrated using quiescent fluids, these studies which show rapid bacterial responses at physiologically relevant hydrogen peroxide levels, suggest that engineered commensal strains may prove beneficial in GI therapies using “smart” probiotics
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
