Caries is a bacterial infection that occurs in dental tissues. Streptococcus mutans is one of the genus Streptococcus with the largest proportion in the natural flora of the oral cavity, and it is also the main pathogenic bacteria of caries. Streptococcus mutans adheres to teeth layer by layer, forming a so-called "biofilm" that is highly resistant to the environment and resistant to antimicrobial treatments. In this study, we designed a genetically engineered bacterium that produces glucanase (DexA)and a phage lyase (ClyR). The former can effectively prevent the formation of biofilm and dissolve the formed biofilm, and the latter can specifically kill Streptococcus mutans. The genetically engineered bacteria prepared in this project have great potential in preventing and treating dental caries.
Figure 1. The principle of genetically engineered bacteria kills Streptococcus mutans.
We inserted the DexA70 and ClyR gene sequences into pUC57 and pBAD expression vectors, respectively. The expression of DexA70 is regulated by the constitutive strong promoter J23101. And the expression of clyR is regulated with an arabinose-inducible promoter araBAD. After the two plasmids were transferred into the same host E. coli, they have the dual functions of destroying the biofilm formed by denatured Streptococcus and being bactericidal, thus providing a more effective solution for the treatment of dental caries.
Figure 2. The map of plasmid. For the construction, we firstly amplified the DexA fragment and pUC57 vector by PCR assay. As indicated in Figure 3, DexA and pUC57 strands were correctly replicated. Then, ligase the DexA fragment and pUC57 carrier to obtain the recombinant plasmid puc57- OmpA-smDexA.
Figure 3. Identification of DexA (1899 bp) and pUC57 (2126 bp) DNA strands by agarose gel electrophoresis.
To identify the recombinant plasmid, we used double enzyme digestion to the puc57- OmpA-smDexA plasmid. PCR was used to verify the monoclonal colony of the strain. As indicated in Figure 4, lines 1 and 2 were positive clones.
Figure 4. Identification of puc57- OmpA-smDexA by agarose gel electrophoresis.
Gene sequencing is used to double verification. As indicated in Figure 5, the returned sequencing comparison results showed that there were no mutations in the ORF region, and the plasmid was successfully constructed. Another plasmid pBAD-Myc-OmpA-ClyR is to be given away by the 2021 iGEM team Shanghai_United_HS. So far, we have successfully obtained the two recombinant plasmids, which can be used to express glucanases and phage lyases proteins.
Figure 5. Sequencing of puc57- OmpA-smDexA
In order to obtain the two proteins smDexA and ClyR, we transferred the recombinant plasmids into E.coli BL21(DE3), expanded the culture in the LB medium, and added IPTG to induce protein expression when the OD600 reached 0.4. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Then we used SDS-PAGE to test the expression of smDexA and ClyR. As shown in Figure 6, the proteins were expressed successfully. At this point, we got the two protein solutions we wanted.
Figure 6. SDS-PAGE assay
Glue diagram of DexA and ClyR proteins. MS is medium supernatant, P is precipitation, and S is supernatant. The molecular weight of DexA protein is 74.33 kDa. The molecular weight of ClyR protein was 30.49 kDa.
To confirm the ability of the DexA and ClyR proteins to inhibit bacterial growth, we used Streptococcus mutans as bacteria for inhibition zone method experiments. As indicated in Figure 7, a transparent circle can be seen around the Oxford cup treated with DexA protein, indicating that the DexA protein can destroy or inhibit the biofilm produced by Streptococcus mutans.
Figure 7. Strain functional test In conclusion, the results of the inhibition zone experiment showed that the protein expressed by the plasmid we constructed could successfully inhibit the growth of Streptococcus mutans, which was in line with the expectations of the project.
We have already collected the figures from our experiments. Combinding DexA and ClyR has the effect of controlling Streptococcus mutans proliferation. By the way, when we compared these data with the positive control group, we surprisingly find that the effect of the DexA+ClyR is better than that.
Because of the effect of the two protein, we believe that if we can use the product in the future and promote it in the market, it will become a great power to against Streptococcus mutans, so as to achieve the purpose of anti-plaque, protect human's teeth, and prevent dental caries.
