Team:MIT/Description

Background

As a chronic but non-lethal condition, dental caries are often overlooked as a target for modern therapeutics, and the quality of dental caries treatment has lagged behind that of more life-threatening diseases such as cancer. However, it is a well established fact that oral health affects overall health--for example, severe cavities can lead to systemic illnesses such as heart disease. Therefore, dental caries is still a prevalent and critical issue.

The process of cariogenesis is highly facilitated by the growth of dense, sticky biofilm on the surface of teeth. This bioflim cuts off the bacteria's access to oxygen, thereby forcing them to revert to anaerobic respiration. Lactic acid is released as a byproduct of bacterial respiration, and leads to demineralization of the enamel and cavity formation.

Biofilm Formation In its natural, non-pathogenic state, S. mutans lives planktonically (free-floating). The transition from the planktonic to biofilm state requires expression of virulence genes, which is coordinated via quorum sensing: a mode of communication employed by many bacteria to coordinate gene expression and synchronous behavior across a population or species. In the case of S. mutans, quorum sensing results in the expression of genes coding for extracellular glucosyltransferase (GTF) enzymes. These enzymes synthesize adhesive glucans, which are used by the bacteria to adhere both to each other and to the enamel of the tooth.

Most quorum-sensing systems involve small, diffusible peptides which are sent out as a signal by one bacteria and received by another. The peptides are detected by a receptor on the membrane or in the cytoplasm of other bacteria, which then initiate a signal transduction pathway that upregulates the quorum-sensing peptide, as well as other genes related to the coordinated behavior of the population. Once enough of the signal has been received by the majority of the population, bacterial behavior shifts to perform a synchronized activity.Biofilm formation is one of the synchronized behaviors initiated by quorum sensing. During this process, S. Mutans create and perpetuate the Competence Stimulating Peptide (CSP), a small molecule that is used to initiate the formation of dental plaque. The pathway through which the cells sense and respond to CSP is referred to the ComCDE pathway.

Quorum Sensing in S. mutans

(hover over each element to learn about its role)

Our Synthetic Circuit

Our goal is to port the ComCDE system into Human Embryonic Kidney (HEK) cells. This would allow our engineered cells to sense the presence of CSP and therefore detect when biofilm formation is about to occur. ComD and ComE will be expressed constitutively, while our three outputs (ScFv's P126 and P136, and Kappa casein) will be expressed under the ComE promoter, which is activated by the ComE response regulator protein as described above.

Outputs

Upon activation of our biofilm-sensing system, our cells will output proteins that have been shown to reduce the biofilm-forming capabilities of S. mutans. Our first protein is kappa casein, a protein derived from bovine milk. It interferes with the adhesive power of the glucans post synthesis through a combination of hydrophilicity and negative charge (Vacca-Smith, 1994). Our second option is using Single Chain Variable Fragments (ScFv's), which are comprised of the variable fragments of monoclonal antibodies joined by a short peptide linker. Our two ScFv's, P126 and P136, bind to the N and C termini of the glucosyltransferases, preventing the production of glucans. (Fukushima, 1992)