Contribution

We utilized 16 promoters from the iGEM Registry to test the impact of different promoters on the expression of LuxR. To achieve this goal, we designed a set of plasmids, where plasmid A is composed of the Promoter library-BBa_B0034-LuxR-BBa_B1006, and plasmid B is composed of pLux-BBa_B0030-mVenusNB-BBa_B1005. By monitoring the changes in the fluorescence intensity of mVenusNB, one can determine the sensitivity of LuxR expression to different promoters.


Add New Documentation to An Existing Part

LuxR and pLux are well-established transcriptional regulatory proteins and their corresponding promoters. In bacteria, once LuxR binds with AHL, the resulting complex associates with the pLux promoter region, thereby initiating the expression of downstream genes. The concentration of AHL is related to the bacterial population density; when it exceeds a certain threshold, the LuxR-AHL complex activates gene expression. pLux is the promoter regulated by LuxR protein, responsible for controlling the expression of downstream genes. When LuxR binds with AHL, the LuxR-AHL complex binds to the pLux promoter, initiating transcription of downstream genes. The pLux promoter regulates the expression of the luxICDABE gene cluster in natural bacterial systems, which is responsible for the production of luminescent proteins.

To enhance the sensitivity of our spoiled milk detection system, we aim to lower the AHL concentration detection threshold to around 5nM. Currently, our system has a half-maximal induction concentration of approximately 11nM, necessitating optimization of the detection pathway. Methods to modify an induction-based transcriptional detection system include adjusting the concentration of the sensor protein, altering the protein-promoter relationship, and mutating the sensor protein. Among these, adjusting the sensor protein concentration is a relatively straightforward approach. We have referenced a study that specifically explored how varying the concentration of LuxR (the sensor protein) affects the final response curve. The study used modeling and experimental methods to reveal that increasing LuxR concentration can significantly reduce the system’s background signal and lower the half-maximal induction curve, providing a new perspective for optimizing our detection system and achieving higher sensitivity and accuracy in practical applications.

Through our experiments, we recorded the maximum response signals of each promoter at different AHL concentrations, providing valuable data to support the subsequent optimization of the system.

Fig. 1 The maximum response signal of the LuxR-pLux genetic circuit with different promoters at different AHL concentrations.

We extracted the maximum fluorescence signals and compared the experimental groups of 0 mM and 50 mM AHL. At a concentration of 50 mM, the induction strengths corresponding to different promoters varied, but all were significantly higher than the control group at 0 mM:

Fig. 2 The significance of the maximum response signal difference between the experimental groups of 0mM and 50mM AHL for the LuxR-pLux genetic circuit with different promoters.

You can find more information at the following links: Parts, BBa_C0062.