Development of a synthetic receptor protein for sensing inflammatory mediators interferon‐γ and tumor necrosis factor‐α

Abstract

Intestinal inflammation has been implicated in a number of diseases, including diabetes, Crohn's disease, and irritable bowel syndrome. Important components of inflammation are interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), which are elevated both on the luminal and submucosal sides of the intestinal epithelial barrier in several diseases. Here, we developed a novel Escherichia coli based detection system for IFN-γ and TNF-α comprised of a chimeric protein and a simple signal transduction construct, which could be deployed on the luminal side of the intestine. OmpA of E. coli was engineered to detect IFN-γ or TNF-α through the replacement of extracellular loops with peptide fragments from OprF of P. aeruginosa. OmpA/OprF chimeras were developed, capable of binding IFN-γ or TNF-α. The specific peptide fragments that bind IFN-γ were identified. IFN-γ or TNF-α binding the OmpA/OprF chimera induced the pspA promoter, driving β-galactosidase production. The OmpA/OprF chimera had a detection limit of 300 pM for IFN-γ and 150 pM for TNF-α. This work will further the development of bacteria based therapeutics for the treatment of inflammatory diseases of the gut.

Using a Redox Modality to Connect Synthetic Biology to Electronics: Hydrogel‐Based Chemo‐Electro Signal Transduction for Molecular Communication

“…Advances in synthetic biology may provide the next generation of biological components for the rapid and cost‐effective processing of chemical information. Synthetic biology constructs could: “recognize” external molecular information; process this information through intracellular signal transduction pathways; and/or generate output responses that can be detected by conventional optical, magnetic, and electrical modalities …”

Connecting Biology to Electronics: Molecular Communication via Redox Modality

“…For example, sequential Ca 2+ ‐alginate codeposition steps generated an electrode coating with the spatially segregated bacterial populations that allows integration of complex cell‐based functionalities . Cell‐based thin film coatings are also important as they can incorporate synthetic biology constructs designed to recognize chemical signals and convert this recognition through intracellular signal transduction pathways into outputs (e.g., redox outputs) that can be measured through device‐compatible modalities (e.g., optical and electrical) …”

“…Technically, small signaling molecules can be detected biologically using bacterial reporter cells that have been genetically engineered to recognize these signaling molecules (e.g., AI‐2) and process this recognition through intracellular gene expression pathways to generate an output response (typically the expression of optically detectable fluorescent proteins) . To enable the communication between molecular modality and electronic modality, we enlist synthetic biology that has sophisticated capabilities to recognize and transduce chemical information . Specifically, we created a synthetic biology (synbio) construct that is capable of transducing AI‐2 recognition into the generation of a reporter enzyme, β‐galactosidase (β‐gal), which can be readily detected optically and electrochemically …”

Fusing Sensor Paradigms to Acquire Chemical Information: An Integrative Role for Smart Biopolymeric Hydrogels

“…The advances in modern biology allow: the production of biology's molecular recognition elements (e.g., enzymes, antibodies and nucleic acids); the design/discovery of entirely new recognition elements (e.g., aptamers and binding peptides); and the application of synthetic biology to access biology's information processing capabilities …”

“…As suggested, the Ca 2+ ‐alginate electrodeposition mechanism is useful for conferring cellular function to sensor coating (note: Ca 2+ ‐alginate hydrogels are routinely used as a matrix for entrapping microbes and a scaffold for tissue engineering). Cell‐based sensing systems are poised to have a significant impact with the development synthetic biology systems capable of detecting chemical signals and converting this recognition through intracellular signal transduction mechanisms to generate outputs that are amplified and/or transduced into device‐compatible modalities (e.g., optical and electrical) …”