Team:UT Dallas/Project

Overview
Harmful bacteria, such as Streptococcus mutans, live in the mouth and convert sucrose into lactic acid and fructose/glucose. The lactic acid creates acidic conditions in the mouth, leading to tooth enamel decay. The fructose/glucose combination forms a sticky polysaccharide called dextran. This molecule is responsible for dental plaque and creates the optimal platform for populous colonies of bacteria on the surface of the teeth. Streptococcus mutans has been determined to be the primary contributor to dental plaque and cavities.
[[File:Streptococcusmutans.jpg]]
Our project entails selectively fighting off this bacteria through 3 different ways:

CscR and FruR
Competence Stimulating Protein, or
Destroying the biofilm upon which the bacteria grows through NspC, Norspermidine.

FruR/CscR
The mouth is very complex in regards to the molecules that enter it. In order to selectively fight S. Mutans, we need to detect its presence in the mouth. One of the ways involved detecting the changing levels of fructose and sucrose. Using operon repressors for both sucrose and fructose, we can detect the concentrations of both of them.

We have chosen two repressors for use in this project:
FruR
CscR

How They Work
The FruR gene encodes a protein which normally binds to and represses the fructose operon. When fructose is present in the system, it binds to the FruR protein and prevents it from repressing the operon promoter.
CscR works in the similar way, but it will bind to sucrose instead.
What We Did
We utilized these repressors, FruR and CscR, to create a construct to detect low levels of sucrose and high levels of fructose. By using the repressor systems, we can detect the formation of plaque and prevent the formation of cavities.

Dex
How It works
Another way to detect S. mutans in the mouth is to detect dextran through dextranase. Dextranase is an enzyme/surface protein that contains a binding domain for Dextran, a very complex, branched polymer of glucans. When Dextranase is on the surface of the bacteria, it binds to Dextran and holds the polymer to the bacteria. We presently believe that Dextranase constitutively works as a surface protein in E. Coli.

What We Did
By using dextranase, we may be able to keep E. Coli in close proximity to S. Mutans, thereby effectively delivering destructive factors to S. Mutans. The dex gene would be included in the E. Coli plasmid to transcribe as a surface protein.

NspC/Norspermidine
How It Works
Norspermidine is a compound that breaks down biofilm by directly interacting with the exopolysaccharid. Norspermidine does this by interacting with charged or polar groups in secondary structure of the exopolysaccharide.

What We Did
Now that we have determined the presence of S. Mutans in the mouth, we can deliver a destructive system to stop bacteria growth. We have determined two ways to accomplish this:
By destroying the biofilm upon which the S. Mutans grows through NspC
By tightly controlling the populations of S. Mutans through CSP and effectively killing all bacteria.
The gene NspC codes for Norspermidine and could be added to our detection systems.

The Com System
How it Works
At normal levels, Competence Stimulating Peptide (CSP) allows a local population of S. Mutans to share plasmids and grow stronger. However, when the concentration of CSP gets too high it activates a genetic pathway that causes some cells to activate cell-death. The quorum signaling system involves five gene products encoded by:
cslAB (comAB)

comCDE
The comC gene encodes a competence-stimulating peptide (CSP) precursor. ComC and ComDE lie adjacent on the chromosome and, together with their gene products, constitute a peptide (CSP)- signaling system including a generating pathway (comC) and a responding pathway (comDE). The other two genes, cslA and cslB, are located in a separate region of the chromosome and encode a CSP-specific secretion apparatus consisting of an ATP-binding cassette (ABC) transporter (ComA) and its accessory protein (ComB), which are involved in the processing and export of the CSP.
What We Did
We attempted to use these quorum sensing molecules which are naturally used by S. Mutans to regulate population growth, competency, and many other uses that are undiscovered. When placed in the mouth, these E. Coli cells will then raise the concentration of CSP and cause the death of any nearby s. mutans cells.