Part:BBa_K1724000

Pcada

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.

Comparing with previous parts about cadmium sensors

Table 1. The comparison with the previous cadmium sensors.Our sensor system is the most sensitive among the sensor mentioned in the table.

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 and 3*10^-8. So we set up the gradient concentration(0, 3*10^-8,10^-7,10^-6,10^-5,10^-4) of the cadmium ion in order to get the pattern of our promoter.

Figure 1. From the left tothe right, the concentration of cadmium chloride is 0,3*10^-8,10^-7,10^-6,10^-5,10^-4 mol/L.We can see that the medium becomes red with the presence of cadmium ion in contrast of the absence of the cadmium. This is the visible test on how different concentration of cadmium ion activates the CadA/MerR operon.

Figure 2. When different concentration of Cd2+ was used to activate our promoter, we detected the fluorescence intensity per OD once every 5 hour. We can easily conclude that when the concentration is lower than 10-8, the promoter wasn’t activated. Followour gradient of concentration we have set,the fluorescence intensity per OD raised obviously every time we measured. We also speculated that the expression strength of promotor became stable when time and the concentration of Cd2+ were all sufficient.

Figure-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^-8. So we set up the gradient of Cd2+ (3*10^-9, 10^-8, 3*10^-8, 5*10^-8) 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 was seen in the picture, when the concentration is 3*10^-9mol/L or 1*10^-8 mol/L the medium didn't change into red but thing goes different when the concentration is 3*10^-8mol/L and 5*10^-8mol/L. The minimum motivating concentration seems between 10^-8mol/L and 3*10^-8 mol/L.

Figure 4. The actual data of activation limit of promoter CadA after having been induced within 21 hours. Lower than 10^-8 mol/L, the cadmium cannot motivate the operon CadA/MerR, contrast of higher concentration. Meanwhile, more than 10^-7mol/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.

Figure 5. The plasmid construction about the device practice.

Contribution of FDR-HB_Peru 2022

In the parts registry of BBa_K1724000, we noticed that iGEM15_SCUT used 0.00003mM, 0.0001mM, 0.001mM, 0.01mM, 0.1mM as their cadmium ions concentration to test their cadmium biosensor with pcadA, a promoter that our team is also aiming to use in the future. Hence, we were curious to see if the E. coli is able to survive at higher concentrations than the ones tested in the registry. Additionally, “E. coli is intrinsically tolerant to high levels of cadmium up to 0.9–1.0 mM” (Qin et al.). Subsequently, we decided to test cadmium resistance of E. coli at final concentrations of 0mM, 0.3mM, 0.6mM, and 1.2mM using the Kirby Bauer method.

From our results, we found that DH5 alpha E. coli was resistant to cadmium concentrations from 0.3 to 0.9mM, as no zones of inhibition were identified. We also found that the bacteria was tolerant to 1.2mM of CdSO4 as well, and small colonies were found. This suggests that E. coli could tolerate cadmium concentrations up to 1.2mM, which is 0.2mM more than what (Qin et al.).suggested. Hence, we were able to conclude that engineered DH5 alpha E. coli could be used at cadmium concentrations up to 1.2mM for cadmium detection and accumulation.

File:Plate.png (figure 1. One of the streaked plates for the experiment)

Works Cited

 Qin, Weitong, et al. Identification of Cadmium Resistance and Adsorption Gene from Escherichia Coli BL21 (DE3) - RSC Advances (RSC Publishing) DOI:10.1039/C7RA10656D. Royal Society of Chemistry, 6 Nov. 2017, pubs.rsc.org/en/content/articlehtml/2017/ra/c7ra10656d.

3. The specificity of the CadA promoter

Figure 6. 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]