Lysine riboswitch is regulated by lysine concentration.When the bacteria colonize the oral cavity, the riboswitch controlled by lysine is turned off. When the bacteria escape the oral environment, the riboswitch opens and expresses sfGFP.The results are shown in Fig.1. The bacteria are in the lysine at a concentration of 30μmol/mL. Fluorescence expression was significantly inhibited, indicating that the lysine riboswitch can work normally.
Fig.1 Relationship between Lysine Concentration and Fluorescence Intensity in Lysine Riboswitch
The toxin RalR acts as a non-specific endonuclease that cuts methylated and unmethylated DNA.
We selected the RalR
amino acid sequence in the TA system derived from E.coli MG1655 for codon optimization and handed it over to
Gen Script for synthesis. Then we imported the sequence into our expression vector pET28a(+) for functional
verification. The engineered strain were streaked into a 1mM IPTG plate for overnight culture and observed that the
engineered strain containing the RalR gene did not grow on the plate. The result is shown in Fig.2;
Fig.2 Safety switch function verification result
It is verified that the expression of the RalR gene has a significant antibacterial effect on our chassis; therefore, we decided to use this gene as the lethal part of our safety switch—toxin protein.