We are the SHSID iGEM team, developing a product that destroys and inhibits pathogenic bacteria in the mouth, thereby preventing and treating dental caries. In order to promote the spirit of iGEM, and inherit and spread the value of iGEM, we specially searched the relevant parts in the iGEM biological parts library and selected BBa_K4030005, the phage lyase clyR. This is the biological section submitted by iGEM21_Shanghai_United_HS in 2021. Our team has fully characterized this part in the laboratory, adding data from identifying the protein expression. This information can provide a good reference for future iGEM teams studying dental caries.
In addition, through literature research, we found another new part dextranase DexA70, which can inhibit the formation of biofilm and destroy the pre-formed biofilm in vitro. We upload its DNA sequence information and basic introduction information in the registry of standard biological parts to provide more choices for inhibiting pathogenic bacteria growth for future iGEM teams.
Dental caries or tooth decay is a common disease, which not only directly affects human oral health, but also often causes adverse symptoms in other parts of the body [1]. According to the global disease statistics in 2016, the incidence rate of dental caries in the population is second only to upper respiratory tract infection, ranking second among common disease. Dental plaque is the primary condition and initiating factor of dental caries. Research shows that the formation of dental plaque is the result of the joint action of many kinds of bacteria. Oral microorganisms based on high-throughput sequencing showed that Streptococcus mutans, lactobacillus, actinomycetes, etc. in plaque were closely related to the occurrence of caries [2-3]. They metabolize and produce various acids, causing the destruction of organic and inorganic substances in teeth to form visible cavities. Effective removal or inhibition of dental plaque formation has become an important means to prevent dental caries [4]. Brushing teeth and flossing are the most basic and practical methods to remove dental plaque, but it is difficult for many people to master and adhere to them. On the other hand, chemical agents such as chlorhexidine or triclosan, which have been used clinically, have significant cytotoxicity in vitro, as well as side effects such as tooth staining. Although fluoride can prevent dental caries by enhancing the acid resistance of teeth, it is difficult to ensure the stability of fluoride concentration. In this sense, the demand for new antibacterial substances from other sources is increasing. Phage lyase is a kind of glycoside hydrolase encoded by phage, which has the function of digesting the cell wall of bacterial host cell. When phage lyase acts on gram-negative bacteria, it shows high bactericidal activity, species specificity and safety in vitro and in vivo, so it has broad application prospects [5]. Among the various bacteriophage lyases that have been found, the chimeric enzyme ClyR (composed of different bacteriophage lyases) has a wide bactericidal spectrum, especially the only bacteriophage lyase that has a strong killing effect on Streptococcus mutans and Streptococcus sanguis [6-7]. These two kinds of streptococcus are the main causes of dental caries.
Figure 1. An example for the uasge of ClyR for debating dental caries.
Figure 2. SDS-PAGE assay
Glue diagram of ClyR proteins. MS is medium supernatant, P is precipitation, and S is supernatant. The molecular weight of ClyR protein was 30.49 kDa.
As can been from figure 2, ClyR was successfully expressed, which set important stage for future engineering.
Biofilm is a highly organized and structured bacterial cell community hidden in the extracellular matrix. Most biofilm substrates are polysaccharides, and dental biofilms are no exception. (1-3) and (1-6) -α-D-glucan polysaccharides are the main components of Streptococcus mutans biofilm matrix. The biofilm produced by Streptococcus mutans is the main obstacle to the effect of various antibacterial agents. The best treatment must be able to eradicate biofilms. Glucanase can inhibit biofilm by hydrolyzing α-1,6-glycosidic bond. Studies have shown that dextranase DexA can inhibit the formation of biofilm and destroy the pre formed biofilm in vitro [6-7].
