Team:SCUT/SensorSystem
Sensor system
Overview
Aiming to detect the cadmium ion as sensitive as possible, we bulit up the sensor system in our engineering E.coli. With the help of synthetic biology technology, we gave the engineering bacterium a dual function: monitoring the amount of cadmium in real time, and adsorbing it if it exceeded a certain level.
After a series of experiments and adequate comparisons, we got the following data and information：
When the cadmium ion concentration was below 3×10-8mol/L, the bacteria would be displayed in green in about 24~48 hours. When the concentration of cadmium ion in the water environment was higher than the detection limit（3×10-8mol/L）, the bacteria would change into red in 3 hours . Meanwhile, cadmium ion would be adsorbed on the surface of the cell.
Since the time that the engineering bacteria cost to turn green was a bit long and it was not so easy for eyes to duly observe a color change, we came to a conclusion that if the bacterium do not turn red within 3 hours, we could assume that the concentration of cadmium ion of the water samples to be tested was below 3×10-8mol/L.
Figure 1: Specific pathways and components mentioned above are shown.
There are two kinds of channel proteins about cadmium ion, including YiiP and ZntA. They transport cadmium iron from extracellular to intracellular.
MerR is an repressor to PcadA. When the cadmium ion concentration is below 10-8 mol/L ,MerR keeps the inhibition of PcadA. Therefore, tetR cannot be activated and the inhibitory effect of tetR on Ptet relieved and it works, then cjBlue expresses. The result is that the engineering bacterium turn green.
Otherwise，the combination of MerR and cadmium ion can relieve the Inhibition effect of PcadA, which makes the downstream gene expressed.
（1）Expression of RFP: the engineering bacterium turn red.
（2）Expression of CsgA-ECs :CsgA-ECs protein is assembled on the surface of the engineering bacterium, and then begins to adsorb cadmium ion.
（3）Expression of TerR:Ptet and the downstream gene is repressed, so that the color of cjBlue vanish gradually.
Introduction
These are the introduction of parts used in the sensor system.
CadA promoter: CadA promoter is sensitive to cadmium ion, which can be repressed by MerR. In high concentration of cadmium ion, the Cd2+ can rapidly bind with MerR and eliminate its inhibition of CadA promoter. In low concentration shows diametrically opposite.
MerR :MerR(wild type),a mercury-sensing regulatory protein, which regulates mercury resistance operons in Gram-negative bacteria, is subjected to directed evolution in an effect to generate a MerR mutant that responds to Cadmium ion but not mercury.[1]That is, the MerR mutant is the cadmium-sensing regulatory protein. To get MerR mutant, oligonucleotide-directed mutagenesis is used to introduce random mutations into the key metal-binding regions of MerR. Finally, Getting the generated Cd-specific MerR mutants appears to be unique.
TetR:TetR family is made up of 47 amino residues. Its structure is helix-turn-helix DNA binding motif and adjacent regions in the three-dimensional structure.[2]TetR can bound to the target operator represses tetA transcription and is released in the presence of tetracycline. In the project, we use tetR as a repressor of Ptet.
YiiP/ZntA: YiiP is a representative member of the cation diffusion facilitator(CDF)family , a class of ubiquitous metal transporters that play an essential role in metal homeostasis. Up to now, a pair of Zn2+/Cd2+—selective binding sites has been localized to two highly conserved as partyly residues(Asp157), each in a 2-fold-symmetry –related transmembrane segment 5 of a YiiP homodimer. Each monomeric subunit contains six transmembrane segments with both N and C terminal located on the cytoplasmic side of the membrane. As partly residues are frequently found in metal binding clusters where they bridge a pair of metal ions by providing two oxygen ligands, each interacting with one of the two adjacent metal ions. [3]So, the YiiP can serve as the channel that binds and cooperative transport cadmium ion.
ZntA is homologous to YiiP. It can translocate the cadmium ion from the outer cell to inner cell that utilizes the free energy derived from the cytosolic ATP.
CjBlue: CjBlue,a chromoprotein from the Cnidopus japonicus sea anemone,naturally exhibits dark green color when expressed.The color isvisibly observed in both LB or on agar plates after 24-48 hours incubating. The sequence is codon optimized for expression in E coli.[4]
Figue 2. Expression of green chromoprotein. Escherichia coli expressing cjBlue (BBa_K592011)by the downstream of promoter tet
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.