We believe that our project can achieve early prevention and early diagnosis of dental caries. The proposed strategy is expected to greatly reduce the prevalence of dental caries globally. We are also aware that we need further improvement in our project to implement our project truly, and it require more time for experimental testing. We have planned for further improvement in this project in future. Unfortunately, due to time constraints, no specific practical tests have been carried out.
In general, our plan is divided into three parts: 1. Prevention; 2. Detection; 3. Safety switch.
Prevention
At present, we have successfully introduced the prevention system into Lactococcus lactis MG1363. However, specific functional tests have not yet been carried out, and further experiments are required for system validation. MG1363 is not an oral probiotic, and further studies are required to introduce the prevention system into the oral probiotic Streptococcus salivarius K12 to prevent dental caries.
Besides, we want to study additional antimicrobial peptide efflux pumps to enhance the tolerance of our chassis to antimicrobial peptides. After improving the tolerance of the chassis to antimicrobial peptides, our chassis can secrete and express antimicrobial peptides to kill dental caries pathogens without affecting the growth and reproduction of microbes in the chassis, making the modified chassis a dominant strain in teeth.
Detection
The experimental results showed that the nlmC promoter has leaked expression. We tried to switch to the nlmAB promoter to reduce the fluorescence leak. However, the sensitivity at this time was also reduced, even affecting the detection effect.
After consulting the literature, it is found that the binding domain of the comE gene exists on the nlmC promoter. Therefore, we plan to modify the nlmC promoter to reduce fluorescence leakage without affecting sensitivity. On the other hand, the two different problems were encountered when we tried the co-cultivation: 1. Some substances excite fluorescence in the BHI medium; 2. The co-cultivation failed to achieve the desired effect.
Therefore, for the problem of medium excitation fluorescence, it may require to optimize the medium or replace it with other fluorescence. The corresponding medium excites the fluorescence weakly and reduces background interference.
For the problem of co-cultivation, there are mainly the following two aspects:
- 1.Escherichia coli is a facultative anaerobe, and Streptococcus mutans is an anaerobe. The two metabolites produced may also interact to a certain extent which is very analytical.
- 2.The presence of other bacteria in the oral cavity may affect the detection effect.
- 3.The sensitivity of our detection system is not enough to achieve the expected severity of caries detection.
Therefore, we will knock out the CSP pathway of Streptococcus mutans and introduce the nlmC promoter or later modified promoter to solve the leakage problem. The addition of fluorescent gene can solve the problem of the cultivation conditions of the engineered bacteria and the detection of bacteria, and may enhaces the sensitivity of the detection system. Besides, we want to optimize the culture conditions according to the characteristics of Streptococcus mutans, so that Streptococcus mutans is the dominant strain while other strains cannot grow and reproduce. The color production by enzymatic hydrolysis requires further testing that is similar to the improvement of fluorescence diagnosis of dental caries severity.
Safety switch
In terms of safety switches, we have verified that the toxic protein RalR has the function of lethal chassis, and the riboswitch can be used to regulate the expression of RalR. However, whether it can inhibit RalR expression in the oral environment, remains to be verified by further studies. We may need to improve the riboswitch system if don’t get good results.
Reference
- 【1】Burton JP, Chilcott CN, Wescombe PA, Tagg JR. Extended Safety Data for the Oral Cavity Probiotic Streptococcus salivarius K12.Probiotics Antimicrob Proteins.2010 Oct;2(3):135-44.
- 【2】Schindler BD, Kaatz GW. Multidrug efflux pumps of Gram-positive bacteria. Drug Resist Updat. 2016 Jul;27:1-13.
- 【3】Hung David C I, Downey Jennifer S, Ayala Eduardo A, et al. Characterization of DNA binding sites of the comE response regulator from Streptococcus mutans.2011, 193(14):3642-52.