Team:SCUT/Basic Part
Basic Part
Basic Part:BBa_K1724000
In 2015, our team tend to apply for the best basic part. Here's the data of our part.
Introduction
CadA promoter is a cadmium-sensitive promoter. Its repressor is MerR. In high concentration of cadmium, the Cd2+ can rapidly bind with MerR and remove its inhibition of CadA promoter. The reverse is the opposition.
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.