From 2007.igem.org

Introduction

In our porject the signal molecular AHL is thought as the electron,so we have to get a method to measuer it.We choose the Detector Cell (BBa_T9002) and fluorescence spectrophotometer.And then,we test our way of detect the AHL and find that our method is useful at a certain extent.The Detector Cell do not work well when the concentration of AHL is high .The fluorescence spectrophotometer result is trust worthly only when the cell density is low.But is is still a good way to measure the 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.
Reaction time for Detecter Cell is 1.5 h.The fluorescence value is measured by Virian Cary Eclipse fluorescence spectrophotometer using the 3ml box at condition : Excitation wavelength-501nm(5nm),Emission wavelength-511nm(5nm),Average time 0.5s.Every sample is measured five times,between which the box was shaked for a better suspending.




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.
Reaction time for Detecter Cell is 2 h.The fluorescence value is measured by Virian Cary Eclipse fluorescence spectrophotometer using the 200ul box at condition : Excitation wavelength-495nm(10nm),Emission wavelength-515nm(10nm),Average time 5s.Every sample is measured five times,between which the box was shaked for a better suspending.

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.
Reaction time for Detecter Cell is 1.5 h.The fluorescence value is measured by Virian Cary Eclipse fluorescence spectrophotometer using the 3ml box at condition : Excitation wavelength-501nm(5nm),Emission wavelength-511nm(5nm),Average time 0.1s.Every sample is measured three times,between which the box was shaked for a better suspending.At each time the sample is measured five times continuously by the instrument.




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)
Reaction time for Detecter Cell is 2 h.The fluorescence value is measured by Virian Cary Eclipse fluorescence spectrophotometer using the 200ul box at condition : Excitation wavelength-495nm(10nm),Emission wavelength-515nm(10nm),Average time 5s.Every sample is measured five times,between which the box was shaked for a better suspending.