Future Directions

Currently, we have characterized our constructs by growing our engineered cells in media buffered to varying levels of pH, and then by measuring fluorescence normalized by optical density over time. Alternatively, we plan to test our constructs by dropping pH over time, as what might occur in the oral environment after mealtimes. By simulating the pH fluctuations in the oral microbiome, we hope to achieve a more accurate representation of the utility of our dual-state promoter in the intended environment.

Ultimately, we envision our dual-state promoters being used to appropriately induce genes that might prevent tooth decay. A variety of genes have been identified the encode proteins and molecules that combat tooth decay.

For example, Tsumori et al. were able to prevent the formation of a dental biofilm by using the mutanase gene, which isolated from Paenibacillus humicus. S. mutans, a major bacterial culprit in the progression of tooth decay, uses glucosyltransferases located on its outer bacterial membrane to synthesize glucan from sucrose. Glucan is a water-insoluble molecule with a high mutan content that S. mutans uses as a link to bind to one another and to the dental surface. In the presence of mutanase, an enzyme that breaks down mutan, Tsumori et al. were able to suppress the formation of S. mutans biofilms.

Alternatively, other plaque bacteria, such as Streptococcus sanguis, Streptococcus milleri, Streptococcus rattus, and Lactobacillus fermentum, have been implicated in returning the oral pH to neutral, or slightly above neutral, through the production of ammonia. These bacteria have developed the arginine deiminase system, which encodes the enzymes arginine deiminase , ornithine carbamoyltransferase, and carbamate kinase, in order to do so. These three main enzymes are used to produce ammonia from arginine found in the diet and saliva. In the future, the arginine deiminase system may be isolated and linked to a dual-state pH-inducible promoter to combat pH fluctuations in the oral microbiome.

MIT’s 2008 iGEM team, Team BiOGURT, succeeded in producing p1025, a small peptide that competitively binds to the same receptors found on the dental surface that S. mutans uses to colonize the mouth. In the presence of p1025, fewer S. mutans cells were able to bind to hydroxyapatite (synthetic teeth).

Any one of these systems, or all three of these systems in series, may be linked to our our dual-state promoters to produce a powerful yet precise system which continually combats oral decay at a steady, tunable basal level, and only fights at elevated levels when this basal level is incapable of maintaining healthy pH levels on its own.