(711c) 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.