Dental-Plaque Material Disintegrator:
The major material that constitutes dental plaque is dextran, a complex branched polysaccharide derived from the condensation of glucose. The polymer main chain consists of α-1,6 glycosidic linkages between glucose monomers, with branches from α-1,3 linkages. In order to decompose this polymer, we utilize dextranase, an enzyme derived from Streptococcus. Dextranase (NCBI Reference Sequence: NP_722336.1) specifically hydrolyzes the α-1,6 glycosidic linkages in dextran and converts the polymer into smaller molecules with higher solubility, leading to easily remove the material.
The dextran in the mouth is synthesized by Streptococcus mutans by series biochemical processes, utilizing Sucrose as raw material. To preclude this process of synthesizing the material of dental plaque, we bring in another enzyme derived from Streptococcus salivarius. FruA, abbreviated from exo-beta-D-fructosidase, can incise the glycoside bond between sucrose molecule, and thus lead to an insufficiency of substrate for S. mutans to utilize.
Expression Plasmid in E. coli:
So as to test the effect of the two enzymes, dextranase and fructosidase, we constructed expression plasmids in E. coli to acquire the proteins and verify the enzyme activity under the experimental condition, stimulating the ph and temperature of human mouth.
Exogenous Nisin-induced Expression Vector:
Strain Lactococcus lactisNZ3900 is a sensitive strain on the concentration of nisin because of its deficiency of immunity gene nisI. Expression of nisl in L. lactis provided the cells with a significant level of protection against exogenously added nisin, indicating that nisI plays a role in the immunity mechanism, also nisC. These genes instruct synthesize proteins could combine with nisin and recede its function as inducement. The original form of NZ3900 has the structural gene nisA, the precursor of Nisin. The gene of nisB helps to correctly fold and “process” the nisin precursor. So, it can produce nisin itself. There are two genes, nisR and nisK, serve to regulate the expression of nisA gene. Gene nisR works as a repressor, specifically bind a site after PnisA. When nisR binds this site, the transcription is locked. While gene nisK can phosphorylate nisR when nisin is specifically reacted with nisK. After the phosphorylation of nisR protein, the repressor is removed and the transcription of target gene can start. So as to appropriately induce the expression of genes with promoter PnisA by exogenous Nisin, we intend to utilize CRISPR/Cas9 system to knock out nisA in the strain NZ3900.
Inducible Lysis-Promoting Elements:
In order to release the proteins synthesized inside the bacteria, a lysis system is needed in this program. iGEM parts BBa_K112000 (T4 holin) and BBa_K112806 (T4 endolysin) was integrated to “self- killing” plasmid, inducing by Nisin, to construct an element of promoting cell lysis, helping to release the intra expression protein (DEX and FruA) and ensure safety.
Safety is always the first thing we concern about. In order to make the two enzymes (DEX and FruA) function on teeth, we want the safest media to contain the enzymes and transport them into oral cavity. After weighing the safety risk of human body and environment, we finally choose yoghurt as the container of our product. Since the yoghurt would be directly taken into human body as the users drink it, we need to judge the safety level of our product extremely carefully.
Considering about the potential risk of our product on human body, we make three special designs to ensure our product being safely edible.
Firstly, we choose Lactococcus. lactis as the bacteria to make yoghurt. As one of the bacterium people use extensively in the production buttermilk and cheese, such as Cheddar and Colby, L. lactis is safe enough to be utilized in our project as the generator of yoghurt.
Secondly, our selection marker is lacF, which can be used on harmless plasmid vector, expressing protein without antibiotic resistance. This means no additional side effect would be presented after people drinking our product.
What’s more, the two enzymes we choose, DEX and FruA, which can decompose sucrose and dextran, are both edible. Thus, the main functional part of the whole design can also be taken into human body without danger, ensuring the safety of our product.
When human body takes the yogurt into digestive system, it is almost unavoidable that some of excreta would be released into environment. Particularly, the genetically-modified bacterias have huge potential risk to influence the environment in an unpredictable way. In order to minimize the effect, we design a self-killing system for those bacteria in our product.
The working mechanism of self-killing system is inducing two enzymes to hydrolyze cytomembrane and cytoderm, killing L. lactis and releasing particular enzymes in the cell. This process is achieved by an inductor called Nisin. As shown above, nisRK serves as corepressor while holin and lysin are hydrolytic enzymes which can split cytomembrane and cytoderm of L. lactis, ensuring no genomic modified bacteria being released to the environment.
As an antibacterial peptide produced by L. lactis, Nisin is commonly used in food industry to preserve cheese, meats, beverages, etc. by suppressing pathogenic bacteria. We don’t need to concern too much about the potential risk to human body of this antibacterial peptide because it is used as a food addictive in everyday life. While the safety of Nisin is justified by empirical experience, it has no inhibitory effect on L. Lactis because a series of immunogene from L. lactis is used to resist the effect of nisin. Judging from this aspect, the inductor of the self-killing system is safe as well.
In conclusion, we design our product in a way that is safe for human body while at the same time having little negative influence on environment as the self-killing system eliminate the further connection between excretory bacterium and the external environment.
Using DEX to cure the dental plaque has been tried by Japanese scientists; they designed to add DEX into dental creams or mouth wash. But the problem for this method was that they cannot ensure the activity of enzymes since the dental creams or mouth wash can hardly provide a “friendly” environment for DEX to work. Basing on this setback, in our design, we add DEX and FruA into dairy product like yogurt in which these enzymes can find suitable ph and temperature, since these enzymes would finally react with dental plaque, also sucrose (FruA), in mouse, where can provide the best fit temperature for enzymes).
For forbidding the further influence of sucrose, our product will not contain sucrose, which is always added in other dairy products for better sweat taste. Instead of that, we would improve our product’s favor by adding Aspartame, a common substitute for sucrose in food product. For this design, our yogurt can also fit the request to the diabetes people, who could not intake too much sugar.
Since we have designed a “self-killing” system, both to kill the L. lactis and to release the inner proteins (DEX and FruA), producer need to add nisin, the compound inducing this system, into yogurt before selling products to consumers, or drinking themselves. An advantage for this design is that our consumers do not need to take any further step to “use” our product.
To find out the most suitable time for our product to work in consumers’ mouth, we have conducted an experiment to test the efficiency of enzymes under “imitate condition”. (Please read detail in our experiment section) Through this experiment, we have got the relation between the utility time and the effect of our enzymes on the actual dental plaque. As a result, we would suggest our consumer to leave yogurt in their mouth for some time, like 0.6 – 1 min, to get the best cure-effect.
Coffey A, Ross RP (2002). "Bacteriophage-resistance systems in dairy starter strains: molecular analysis to application". Antonie van Leeuwenhoek. 82 (1–4): 303–21. doi:10.1023/A:1020639717181. PMID 12369198.
Ryan, Lisa (12 January 2016). "'Magic MOLD' could wipe out two lethal conditions: Natural preservative that grows on dairy products 'kills cancer cells and antibiotic-resistant bacteria'".
Daily Mail. Retrieved 14 January 2016.