Towards a Genetically-Engineered Bacterium for Gastrointestinal Wound Healing

Bacteria are often thought of as “good” bacteria (e.g., commensals, probiotics) or “bad” bacteria (e.g., pathogens). Synthetic biology enables the augmentation of biosynthetic capabilities and retooling of regulatory structures in the creation of cells with unprecedented ability to make products. One can also, however, think of the cell as the product – a cell that operates in a noisy environment to execute non-native tasks. There have been several recent reports of the rewiring of bacterial cells to function as conveyors of therapeutics. The engineering and rewiring of the bacteria such as E. coli into ‘smart' bacteria potentially allows for a broad range of applications, from the treatment of wounds, the elimination of pathogenic strains, to the delivery of vaccines, particularly in the GI tract.

‘Smart' bacteria must survey their environs, respond to the appropriate cues, while at the same time not respond to inappropriate cues. They must stay on task. Our approach comprises electrochemistry and synthetic biology for the creation of a biological ‘test track' for ensuring the appropriate design and testing of engineered bacteria. We are engineering bacterial motility for response to wound-generating signals such as hydrogen peroxide and reactive oxygen species. Specifically, we have placed motility enzyme CheZ under the control of the hydrogen-peroxide-responsive oxyR promoter so that the “run” in the tumble and run scheme of bacterial movement is externally regulated. Engineered cells exploit pseudotaxis for directional swimming.

In addition, we engineer the surface of these bacteria for the display of therapeutic agents including human proteins and enzymes. Specifically, we have expressed human tissue transglutaminase (htTG) on the outer surface of E. coli for the crosslinking of proteins containing exposed lysine and glutamine residues. We exploit the AIDA (AIDA-1) system for presentation of the htTG on the outer surface using the motives shown below. For therapeutic application, tissue transglutaminase would then potentially aid in wound repair. Our presentation will highlight both successes in engineering “smart” bacteria using this methodology as well as limitations based on system design and the parameter space for use.