(164t) Leveraging Synthetic Biology and Gut-on-a-Chip Systems to Investigate the Mechanistic Role of H2s in the Gut

The human gut microbiota is a complex biological system composed of trillions of bacteria and their interactions with the host. Hydrogen sulfide (H₂S), a metabolite of various gut microbes, has been correlated with diseases such as IBD and colorectal cancer. However, H₂S has also been shown to protect tissue from oxidative stress and act as a signaling molecule. Past studies have investigated H₂S effects on gut epithelial cells but were done in static cultures and H₂S was not microbially derived. Traditionally, static epithelial cultures and animal models are used to study the gut but are plagued by low transcriptomic similarity, high drug translation failure rates, and ethical issues. In vitro flow systems, such as the gut-on-a-chip (GoC), designed with human-derived tissue are easier to probe than animal models and more genetically representative than static cultures. We hypothesized the effects of H₂S on gut epithelial cells could be concentration-dependent and more accurately investigated in a GoC system. Researchers estimate H₂S levels in the human gut vary from 0.2 to 3 mM. To investigate our hypothesis, we engineered the bacterium Escherichia coli for tunable production of H₂S via titratable expression of l-cysteine desulfidase, which catalyzes the conversion of l-cysteine to H₂S. Since E. coli has various native enzymes with l-cysteine desulfidase activity which could limit our ability to explore lower biologically relevant H₂S levels, we first targeted four native E. coli genes for deletion via the CRISPR-Cas9 system, resulting in a strain with very low background sulfide production. We then assessed desulfidases from Fusobacterium nucleatum and the native E. coli yhaOM operon which we expressed in plasmids with varying promoter strengths. Experiments in Hungate tubes confirmed our ability to vary H₂S levels from 0.025 to 1 mM. Next, we introduced our strains into a GoC system and showed we can tune H₂S levels from 0.2 to 1.5 mM for up to 72 hours. Studies are underway using this system to investigate how different concentrations of H₂S affect gut permeability, monolayer integrity, and genetic expression. It is postulated H₂S induces cell cycle arrest and DNA damage which we will investigate with qRT-PCR of the epithelial cells. Through our work, we aim to answer fundamental questions about H₂S role in the human gut and showcase a platform for integrating synthetic biology and GoC systems to study gut metabolites with ambiguous roles in disease.