Part:BBa_K4619012
QS-LuxRm-PluxR
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 789
Illegal NheI site found at 812
Illegal NheI site found at 895
Illegal NheI site found at 918 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 412
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 823
Illegal SapI.rc site found at 962
Quorum sensing is used by a large variety of bacteria to regulate gene expression in a cell-density-dependent manner.
Bacteria can synchronize population behavior using small molecules called autoinducers that are produced by cognate synthases and recognized by specific receptors. Quorum sensing plays a critical role in regulating diverse cellular functions in bacteria, including bioluminescence and so on.
The system is used to detect and express upstream signals without using positive feedback. The autoinducers (AHL), produced by the upstream system, can diffuse through the cell membrane into the growth medium. Here, we used a mutated LuxR–lux box system, called LuxRm-Pluxm. Pluxm promotor can not interact with wild-type LuxR protein. Compared with the wild-type LuxR–lux box system, the transcriptional activities of LuxRm-Pluxm are better, the background expression is more tightly regulated.
The J23119 promoter drives the expression of the luxRm gene continuously. If the upstream pathway is closed, AHL concentration is low, only showing slight leakage. As the upstream pathway opens, the autoinducers in the medium start to accumulate in a confined environment. When enough AHLs have accumulated in the medium, they enter the cell where they directly bind the LuxRm protein to activate msfGFP expression. High levels of AHLs activate the msfGFP expression, possessing a small volume and impressive performance simultaneously. A high intensity of light can be detected.
Remark: we use msfGFP as a convenient reporter in our experiments, the Nanoluc showing in the diagram refers to our design
Experiment & Result
Here, we designed AHL-induced quorum sensing systems without a positive feedback system and compared the relative fluorescence intensity and OD value of colonies after induction for 6 hours by adding the correct AHL (VAI) and the wrong AHL (PAI). For the AHL sensitivity measurements, the E. coli containing the plasmid was cultured in LB medium until OD600 = 0.5 – 1.0 and then induced by correct AHL (VAI) and the wrong AHL (PAI) dissolved in DMSO medium. For fluorescence determinations, 200 µl aliquots of each sample were added to a 96-well plate for 4 hours. Fluorescence spectra were recorded using a fluorescence spectrometer at 485 and 528 nm for excitation and emission wavelengths, respectively.
We divided the difference in fluorescence intensity before and after induction by the OD value of the colony to obtain the relative fluorescence intensity of the unit colony; the graphs are as follows:
As can be seen from the above figure, after four hours of induction, the induction effect of adding AHL that matches the system is better than that of adding mismatched AHL, indicating that our system is not susceptible to interference and the promoter has specificity;
We also measured the fluorescence value change curve at a concentration of 10nM. As shown in the figure above, at a concentration of 10nM, a certain induction effect has been produced. However, the relative fluorescence change intensity is still not obvious enough, and further experimental conditions and content are needed.
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