Team:SCUT/Inthelab
In the lab
1.Promoter CadA quantification
In 2015 SCUT team, we expressed our three proteins by the downstream of promoter CadA. Promoter CadA is Cd-activated promoter with the presence of MerR. When enough Cadmium existed, the gene downstream can be express. We construct MerR/CadA operon and place the RFP behind the CadA promoter. As the detection limitation mentioned below, the lowest concentration of the cadmium ion is between 10^-8 mol/L and 3*10^-8 mol/L. So we set up the gradient concentration(0, 3*10^-8,10^-7,10^-6,10^-5,10^-4 mol/L) of the cadmium ion in order to get the pattern of our promoter.
Figure 1: From left and right the concentration of cadmium chloride which is added into medium is 0,3*10^-8,10^-7,10^-6,10^-5,10^-4 mol/L. We can see that the medium become red with the presence of cadmium ion in contrast of the absence of the cadmium. This is the visible test how different concentration of cadmium ion activates the CadA/MerR operon.
Figure 2. Accumulation of RFP fluorescence in culture of engineering E.coli.The promoter CadA Strength test in the concentration of 10^-6 mol/L and 0mol/L cadmium chloride and 0mol/L. We construct Constructive promoter +MerR+termintor & PCadA+CsgA-EC(n)+RFP+tetR+termintor into E.coli. The abscissa is the time after we add the cadmium chloride . The ordinate is RLU/OD. The curve indicates that the engineering E.coli can be activated and go into stabilization phase at 9 hours.
Figure 2-2. The brief introduction of plasmid of the CadA promoter quantification.
2.Detection limit of promoter CadA
In order to test the detection limit of the promoter CadA, we first set up gradient concentration of Cadmium（10^-8,10^-7,10^-6,10^-5,10^-4mol/L）to estimate the probably range. As the result mentioned above, the promoter CadA can be activated over the concentration of 3*10^-8mol/L. So we set up the gradient of Cd2+ (3*10^-9, 10^-8, 3*10^-8, 5*10^-8mol/L) and obtain the detection limit of promoter CadA.
Figure 3. The visible test of detection limitation. The flag on the 50ml tube was the concentration of cadmium ion. As seen in the picture, 3*10^-9 mol/L and 1*10^-8 mol/L Cd2+ cadmium medium didn't change into red contrast of 3*10^-8 mol/L and 5*10^-8mol/L. The minimum motivating concentration is between 10^-8mol/L and 3*10^-8 mol/L.
Figure 4. The actual data of activation limit of promoter CadA after inducing within 21 hours. Lower than 10^-8 mol/L, the cadmium cannot motivate the operon CadA/MerR, contrast of the higher concentration. Meanwhile, more than 10^-7 mol/L, the expression of RFP stays steady, the maximum is near 11000 RLU/OD.
So we can come to a conclusion that the lowest concentration of cadmium to activate promoter CadA is between 10^-8 and 3*10^-8 mol/L.
3. The specificity of the CadA promoter
Figure 5. The specificity of the MerR/CadA promoter. As seen in the picture, the medium added cadmium in became red, in contrast that the medium which was added nothing or added other heavy metal maintained the same situation.
3. The specificity of the CadA promoter
After finishing the detection of The promoter of quantification, we started to test whether our device could work on. We first incubated our engineered E.coli wiht different strength EC in LB medium. After 48-hour-culture, we started adding various concentration of cadmium ion(10^-4,10^-5,10^-6,0 mol/L) and reporting the change of the cadmium ion and color.
Figure 6. The actual process after adding cadmium chloride. We added the cadmium ion before the 48 hours incubation of our engineering E.coli. From left to right, the sample added the same concentration of cadmium(10^-6mol/L) was taken in 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours and 12 hours. We could see the color change as time went on(from green to red) so as to demonstrate that our device worked.
Figure 6-2. The plasmid construction about the device practice.
Figure 7. The capacity of EC adsorption. The abscissa is the initial concentration of cadmium ion before treating. The ordinate is concentration of cadmium chloride. As seen in the picture, A,B,C,D,E means in different original [Cd2+] the treatment data of EC which can adsorb the cadmium efficiently because the concentration of cadmium in EC-treated medium is one-third (or half) less than the control.