Poor oral health (tooth decay and infection) can lead to many painful symptoms and complicated treatments. Mouth bacteria are a primary culprit in tooth decay, as lactic acid secreted by natural bacteria in the human mouth decreases pH, leading to cavity formation. The enamel demineralization threshold is known to be around pH 5.5. The overall vision of our project is to prevent mouth pH from falling below 5.5 by engineering bacteria to sense pH and respond by increasing the alkalinity. To do this, we aimed to create genetic pH sensing elements that would work in the mouth.

Our project laid the foundation for a dual-state promoter consisting of two promoters in series: a constitutive promoter active at a (tunable) basal level and an upstream, pH-inducible promoter. Previous research has found that the E. coli genes Asr and GadA are highly expressed when pH drops below 5.5. This research drove us to choose the promoter elements for GadA and Asr as our pH-inducible promoters.

We have characterized a new pH-responsive promoter (Asr) as well as an iGEM-characterized promoter (GadA) and a constitutive promoter (Lpp). Ongoing work will evaluate how promoters in series affect each other and further characterize the dual state phenomenon. Here we used E. coli for the ease at which it is genetically manipulated to provide proof of principle. Additionally, we tested these promoters with GFP for an easy visual output. Ultimately the aim is to engineer bacteria native to the oral microbiome to sense and respond to drops in pH. We envision that these bacteria will induce enzymes that catalyze reactions to counteract the decrease in pH, preventing the demineralization of the tooth.

Summary of Problem

Following meals, the pH within the mouth drops substantially which leads to cavities.

Dual State Promoter

In order for a quick response from basal level, we designed the dual state promoter.