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constitutive promoter+RhlR protein generator(LVA-)+RhlR/PAI2 Inducible promoter+RBS(B0030)+RFP+Terminator The max excitation wavelength for the RFP we used is 584nm,and the max emission wavelength is 607nm. We constructed an amplification plasmid,containg RhlR protein(LVA-) and RhlR/PAI2 Inducible promoter with RFP. Then we transfected the plamid to Trans 5α cells, and used the cells to do follow experiments.

FIG.1 The cell was cultured in LB culture and shaked at 37℃ for 12 hours. Then we added the PAI1 and incubated the cell for another 6 hours. We set 18 samples in parallel , and calculated their average, the results of the experiment are shown.

FIG.2 The cell was cultured in LB culture and shaked at 37℃ for 12 hours. Then we added the PAI1 and incubated the cell for another 6 hours. We set 8 samples in parallel each experiment ,and calculated their average, the results of the experiment are shown. From the above data and graph,We can see that with the increase of PAI1’s concentration, fluorescence intensity also gradually increased.And at 200umol, the fluorescence intensity reached peak. Still, how the Fluorescense Intensity/OD changes with the dose of PAI1 over 200umol changes needs further test.

FIG.3 The cell was cultured in LB culture and shaked at 37℃ for 9 hours. Then we added the PAI1 and incubated the cell for another 8 hours. We set 18 samples in parallel ,and calculated their average, the results of the experiment are shown.

FIG.4 The cell was cultured in LB culture and shaked at 37℃ for 12 hours. Then we added the PAI1 to the final concentration 200umol.We set 6 samples in parallel ,and calculated their average, the results of the experiment are shown.

FIG.5 The cell was cultured in LB culture and shaked at 37℃ for 9 hours.Then we added the PAI1 to the final concentration 200umol.We set 18 samples in parallel ,and calculated their average,the results of the experiment are shown. It’s obvious that with the increase of time, fluorescence intensity also increased gradually. These data all above provide evidence that autoinducer molecules PAI1 could enter the cell and induce the expression of RhlR/PAI2 Inducible promoter.
constitutive promoter+RhlR protein generator(LVA+)+RhlR/PAI2 Inducible promoter+RBS(B0030)+RFP+Terminator The difference between this amplification plasmid we constructed and the one above is the existing of LVA.

FIG.6 The cell was cultured in LB culture and shaked at 37℃ for 12 hours. Then we added the PAI1 and incubated the cell for another 6 hours. We set 18 samples in parallel , and calculated their average, the results of the experiment are shown. We can see the fluoresence intensity is small at each dose. This is because with the existence of LVA’s ,the half-life period of RhlR protein is too short to combine with PAI1.Hence,the promoter cann’t be induced properly. From the data,we can confirm that the autoinducer molecules PAI1 could enter the cell and combine with RhlR protein,and then induce the expression of RhlR/PAI2 Inducible promoter.
constitutive production of LasR+LasR/PAI1 Inducible promoter +RBS(B0030)+GFP+Terminator The max excitation wavelength for the GFP we used is 501nm,and the max emission wavelength is 511nm. It’s another amplification plasmid we constructed.And we planed to use the two plasmids to respond to different sensors.

FIG.7 The cell was cultured in LB culture and shaked at 37℃ for 9 hours.Then we added the PAI1 and incubated the cell for another 8 hours.We set 8 samples in parallel for each dose ,and calculated their average,the results of the experiment are shown.
2.Time Gradient

FIG.8 The cell was cultured in LB culture and shaked at 37℃ for 9 hours.Then we added the PAI1 to the final concentration 200umol.We set 6 samples in parallel at each time ,and calculated their average,the results of the experiment are shown.