Diagnostic and Therapeutic Microbial Circuit with Application to Intestinal Inflammation

Area of Science:

• Synthetic Biology
• Microbiome Engineering
• Genetic Engineering

Background:

• Engineered microbes offer therapeutic and diagnostic potential within the human microbiome.
• Current limitations include single-input responses, hindering precision and effectiveness.
• Complex conditions like inflammatory bowel disease require multi-input microbial responses.

Purpose of the Study:

• To engineer a probiotic Escherichia coli strain Nissle 1917 (EcN) with an AND logic gate for multi-stimuli response.
• To enable conditional therapeutic delivery based on specific biological and external signals.
• To demonstrate the secretion of a therapeutic cytokine, IL-22, for potential inflammatory bowel disease treatment.

Main Methods:

• Implementation of a split activator AND logic gate in E. coli Nissle 1917.
• Utilized tetrathionate (inflammatory biomarker) and anhydrotetracycline (aTc) as dual input signals.
• Modeled AND gate dynamics using chemical reaction networks and performed in silico parameter tuning.
• Engineered the system to secrete IL-22 via the hemolysin secretion pathway.
• Validated therapeutic cytokine production in a germ-free transwell model of human gut epithelium.

Main Results:

• Achieved 4-6 fold induction of the AND gate with minimal leak when both signals were present.
• In silico modeling identified critical parameters for circuit selectivity.
• Engineered EcN successfully secreted IL-22, eliciting comparable host cytokine responses to recombinant IL-22 in vitro.
• Demonstrated minimal off-target effects in a gut epithelial context.

Conclusions:

• Developed a scalable workflow for engineering cytokine-secreting microbes using logical signal processing.
• Successfully created a probiotic E. coli strain capable of sensing and responding to multiple environmental cues.
• This engineered microbe demonstrates feasibility for targeted IL-22 delivery, offering a promising strategy for inflammatory bowel disease management.