From 2007.igem.org

Introduction

In our project the flow of signal molecular AHL is considered as the current,so we have to design a method to measure it.We choose the Detector Cell (BBa_T9002) which could accept AHL to produce Green Fluorescence Protein and fluorescence spectrophotometer which could quantificationally measure the fluorescence intensity.And then,we test our way of detecting the AHL to find that our method is valid within a certain concentration range of AHL. The Detector Cell do not work well when the concentration of AHL is high .The fluorescence spectrophotometer result is trustable only when the cell density is low. But it is still a good way to determine the concentration of AHL .

Results

Figure 1: Regardless of the cell concentration, production of GFP (Represented by the intensity of fluorescence) has a linear relationship with the concentration of AHL within the range of 0.01-1 uM.

After mixing the detector cells culture and the AHL solution for 1.5 hours, the fluorescence value is measured by Virian Cary Eclipse fluorescence spectrophotometer using the 3ml cup at the following condition : Excitation wavelength-501nm(5nm),Emission wavelength-511nm(5nm),Average time 0.5s. Five individual parallel experiment for each sample have been execurated to get a more accurate result.




Figure 2: The production of GFP is initialized when the concentration of AHL reaches to 0.01uM, then keep increasing linearly within the range of 0.01-1um, finally varies unconspicuously at values over 1um.

After mixing the detector cells culture and the AHL solution for 1.5 hours, the fluorescence value is measured by Virian Cary Eclipse fluorescence spectrophotometer using the 200ul cup at the following condition : Excitation wavelength-495nm(10nm),Emission wavelength-515nm(10nm),Average time 5s. Five individual parallel experiment for each sample have been execurated to get a more accurate result.

Figure 3: The dependability of this measuring method is tested by calculating the standard deviation of datas from three parallel experiments. As shown on the graph, the length of vertical lines display the value of standard deviation at a particular concentration of AHL, so the denser AHL is, the more inaccuracy the data could be.

After mixing the detector cells culture and the AHL solution for 1.5 hours, the fluorescence value is measured by Virian Cary Eclipse fluorescence spectrophotometer using the 3ml cup at the following condition : Excitation wavelength-501nm(5nm),Emission wavelength-511nm(5nm),Average time 0.5s. Three individual parallel experiment for each sample have been execurated to get a more accurate result.

Figure 4: Relationship between the cell density and the expression of GFP. Obviously, GFP increased with the density of E.coli cells.The abscissa means the relative od(which is measured under diluting).When the cells reach a high density,the fluorescence value will not increase at the same rate as before.We guess that it is because some cell was covered at a high density.Another result ,doen using the 3ml box ,which is not presentd here shows the same result that when the cell od is high than 0.8 the fluorescence value is not direct ratio to the cell density.(we guess that it is because the box is bigger)

After mixing the detector cells culture and the AHL solution for 2 hours, the fluorescence value is measured by Virian Cary Eclipse fluorescence spectrophotometer using the 200ul cup at the following condition : Excitation wavelength-495nm(10nm),Emission wavelength-515nm(10nm),Average time 5s. Five individual parallel experiment for each sample have been execurated to get a more accurate result.