Compared to the old part projects BBa_K4030009， which is a biological part submitted by iGEM21_Shanghai_United_HS in 2021, the ClyR is under araBAD promoter, which is induced by arabinose. And ClyR is linked with ompA and this sequence is inserted into a plasmid. The constructed plasmid exhibited weak activity. In this part, we selected the dextranase DexA. DexA can inhibit the formation of biofilm and destroy the pre-formed biofilm dextranase DexA in vitro. We designed a genetically engineered bacterium that produces a glucanase DexA and a phage lyase ClyR. The former can effectively prevent the formation of biofilm and dissolve the formed biofilm, while the latter can specifically kill Streptococcus mutans. The genetically engineered bacteria prepared in this project have great potential in the prevention and treatment of dental caries.
Figure 1. The principle of genetically engineered bacteria kills Streptococcus mutans.
In order to obtain the two proteins smDexA and ClyR, we constructed and 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 2, the proteins were expressed successfully. At this point, we got the two protein solutions we wanted.
Figure 2. 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 3, 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 3. 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.
