Results

1. Construction of the Biosensor plasmids

We design two kinds of biosensor to detect the heavy metal cadmium, zinc and arsenic. For arsenic, our project used the ArsD, ArsA, ArsR promotor fused amilGFP being its reporter protein. For cadmium and zinc, we utilize the operon CadA to expresses reporter feruloyl esterase in Bacillus subtilis.

The first series of plasmids pUC57-ArsD-amilGFP, pUC57-ArsA-amilGFP, and pUC57-ArsR-amilGFP were a gift from 2021iGEM Team Shanghai_United. The second series plasmid pHY300PLK-PcadA-Biosensor was handed over to GenScript for synthesis. The synthesis report is as follows.

Based on the plasmid pHY300PLK-PcadA-Biosensor, we designed a plasmid pHY300PLK-Pveg-Biosensor for constitutive expression feruloyl esterase in Bacillus subtilis. The difference between them is that the cadmium ion-inducible promoter PcadA is replaced by a constitutive promoter Pveg.

Figure1. Gel electrophoresis results Veg fragment and pHY300PLK vector.
A. Lane M DNA ladder; Lane1, 2 Veg fragments, 97bp, correct;
B. Lane M DNA ladder; tLane1 pHY300PLK vector, 5902bp, correct.

To construct the plasmid pHY300PLK-Pveg-Biosensor, the promoter Veg fragments and pHY300PLK vector were amplified by PCR, respectively. As can be seen from the figure 1A, there was a specific band at the position of 97bp, indicating that the PCR amplification of the veg fragment was successful. And the pHY300PLK vector was also amplified successfully (figure 1B).

The DNA fragment Veg and pHY300PLK vector ligate using the Gibson assembly method. Then, the recombinant plasmid was transformed into the competent cells DH10b. The bacteria spread to the selection plate and incubated at 37 ℃ overnight.

Figure 2. The plate of recombinant plasmid pHY300PLK-Pveg-Biosensor.

We picked up 15 colonies for performing colony PCR. The M representative DNA ladder, lane 1-16 representative colonies. The result showed all selected colonies have the correct band. Thus, we sent colonies No. 1, 3, 5, and 7 to DNA sequencing.

Figure 3. colony PCR

The sequence alignment showed that there is no mutation or mismatch. Thus, we choose colony No.3 for subsequent assay.

Figure4. Sequence alignment

2. Functional Test

2.1 Arsenic-biosensor

We have tested the ability of three biosensors for detecting the heavy metal arsenic. The plasmids were transformed into E. coli and tested while in LB medium. The florescence intensity to reflect the GFP concentration after the induction of arsenic ions in different concentration. Three promoters are involved in the test and comparison: ArsA, ArsD, and ArsR. Under different time scales, the fluorescence intensities of those promoters are shown in the following graphs:

Figure 5. GFP intensity in different concentaiton of As and cultivation time
The 0-hour data set as the control group.

There was no significant expression of GFP displayed at 1 hour regarding all three types of biosensor. This result indicates that under the time period of 1 hour, the three biosensors are not able to induce the expression of GFP although the medium contain increasing arsenic solutions concentration. However, starting from 2 hours, the data demonstrated certain level of positive association amount the florescence intensity and the arsenic concentration ranging from 10ug/L to 50ug/L. The higher concentration of arsenic might inhibit the growth of bacteria, so the GFP intensity decreased. No remarkable difference of florescence intensity was detected between 2 hours and 3 hours. At 2 hours, 50ug/L is the maximum florescence expression for ArsA and ArsD, and for ArsR it was about 20ug/L.

In conclusion, the results of the fluorescence data show that our arsenic detection sensor can reflect the arsenic ion concentration in the sample during the reaction period of 2 hours and the detection concentration range from 0 to 100μg/L, which is in line with the expectations of the project design.

2.2 The Activity of Feruloyl Esterase expressed by promoter veg

In order to confirm the protein expression and secret into the culture medium, the plasmid pHY300PLK-Pveg-Biosensor which can consistently express feruloyl esterase(FAE) was transformed into Bacillus subtilis and contand tested in different cultured time. Because FAE catalyzes the decomposition of the substrate methyl ferulate, and the enzyme activity of FAE can be obtained by detecting the decline rate of methyl ferulate at 340 nm. The chemical equation is as follow:

feruloyl-polysaccharide + H2O = ferulate + polysaccharide

Figure 6. The activity of ferulyol esterase in different culture time.

The results showed that the enzymatic activity of FAE increased with the prolongation of culture time, indicating that the expression of enzyme increased with the increase of culture time. At the same time, since we detected the enzymatic activity of the protein in the supernatant of the culture medium, this shows that our protein can indeed be expressed in Bacillus subtilis and secreted to the outside of the cell, which verifies that our design idea is correct. It has laid a solid foundation for the cadmium-inducible promoter(Pcad) and the detection of cadmium ion concentration.

2.3 Cadmium-biosensor

We tested the ability of cadmium-biosensor for detecting the heavy metal cadmium. The plasmid pHY300PLK-PcadA-Biosensor was transformed into Bacillus subtilis(Fig7.) and tested while in different concentration of cadmium nitrate. The enzyme activity of feruloyl esterase(FAE) is to reflect the cadmium concentration after the induction of cadmium ions. Under different cadmium concentration scales, the enzyme activity of FAE is shown in the figure 8.

Figure 7. Plate of the transformants of pHY300PLK-PcadA-Biosensor/Bacillus subtilis.

Figure 8. The activity of Ferulyol Esterase in different concentaiton of Cd. The data were collected in 0.5h.

As shown in the graph, the more the cadmium is, the more enzyme activity it has. While, when the concentration of cadmium reaches 50μg/L, its enzyme activity value tends to be stable at about 0.35. We speculate that when the cadmium ion concentration exceeds 50μg/L, the host Bacillus subtilis cannot express the protein normally, so the measured enzyme activity value is flat. This indicates that our current cadmium biosensor is suitable for the detection of samples with cadmium ion concentration in the range of 0-50μg/L.