PnlmC (BBa_K1510002) is derived from Streptococcus mutans UA159. The intracellular response regulator protein comE can activate PnlmC. The activation of comE requires contact with the activated histidine kinase receptor membrane protein comD and sufficient Capability stimulating peptide (CSP). Therefore, the main function of the promoter is to sense the quorum sensing substance—the ability stimulating peptide (CSP).
In 2014, the NYMU-Taipei team cited the relevant data of the article ComCED signal loop precisely regulates nlmC expression in Streptococcus mutans published by Liu, T., et al. in Annals of Microbiology, but did not characterize it.
This year, we tested the part in conjunction with our project.
The recombinant strain E.coli BL21 (DE3) with pET28a-DEG vector was as the detector. Different concentrate CSP was added into the culture medium. When the concentration of CSP reached 0.5mg/ml, the comD-CSP complex would Phosphorylation of comE, phosphorylated comE and nlmC promoter combined to start the expression of the downstream fluorescent gene GFP (Fig.1). The fluorescence intensity increased gradually as the culture time prolonged. After 24h, there was a significant difference in fluorescence intensity. This result showed that the quorum sensing signaling system in E.coli detector we constructed was functional.
Figure 1. The relationship between CSP concentration, time, and fluorescence intensity
Figure 2. Comparison of fluorescence values of samples without CSP induction
Figure 2 shows the comparison with the control group, certain fluorescence intensity was measured in the engineering bacteria containing the nlmC promoter, indicating that the nlmC promoter has fluorescence leakage. In the future, we shall try to redesign or mutate a new low-leakage promoter based on the nlmC promoter and comE binding domain to achieve high sensitivity and low detection limit detection.