Team:ASTWS-China/Results

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Results

Copper detection device 1.0 (BBa_K2826008)

This part is an improvement of part BBa_K2088015, which consisted by the same copper ion detecting promoter and GFP as reporter. Comparing with BBa_K2088015, our improvement is using RFP to replace GFP as reporter.

As shown in Figure 1, with the extension of culture time, the fluorescence intensity of eRFP gradually increased which indicates that the expression level of eRFP in the medium gradually increased. In other words, the copper detection function of this working system was working normally. The concentration of copper ions in the waste water is close to 3ml/L. The detection results are similar to measurement result of ICP. Besides, as shown in Figure 2, as the copper concentration increased, the color turned redder. It is proved that the copper detection function of this working system was working as we expected.

Additionally, we also used this device to sense copper concentration of the real sewage sample (Volume medium : Volume sewage = 1:1). As shown in Figure 1 and 2, the curve of waste water was coincided with the one of 3 mg/L copper concentration. And the comparative photos was revealed the same truth. In the other words, the copper concentration of waste water sensing by our detection device was nearly 6 mg/L which is approximative to 5.75 mg/L. If further to optimize, our device could be a promising method to sense copper pollution in real environment.

Figure 1: Curve of REP fluorescence intensity and culture time under different copper concentrations

Figure 2: E. Coli DH5α after culturing for 10 hours under different copper concentrations

Copper detection device 2.0 (BBa_K2826009)

As shown in Figure 3, we can see that copper detection device 2.0 can detect the copper ion concentration in the solution by the red fluorescence intensity. However, the logic reluctance components (BBa_C0040+BBa_R0040) in this device not work. As the copper ion concentration increased, the expression of GFP is not well prevented, and this part need to be improved (Figure 4 and 5 ).

Figure 3: Curve of RFP fluorescence intensity and culture time under different copper concentrations of E.coli containing BBa_K2826009

Figure 4: Curve of GFP fluorescence intensity and culture time under different copper concentrations of E.coli containing BBa_K2826009

Figure 5: E. Coli DH5α containg BBa_K2826009 after culturing for 10 hours under different copper concentrations

Copper Treatment device 1.0 (BBa_K2826011)

Plasmid cleavage and DNA sequencing data showed that the MbnABC gene was successfully ligated in BBa_K2826011. After culturing for 10 hours in LB media, the strong expression of RFP was observed, and it was indicated that MbnABC was also expressed.

Figure 6: E. Coli DH5α containg BBa_K2826011 after culturing for 10 hours

Copper Treatment device 2.0 (BBa_K2826013)

According to codon preference, MbnABC was redesigned and synthesized so that it can be expressed efficiently in E.coli.

And a copper ion binding carrier: cop.A2 (BBa_K2826013) was constructed, which was composed of BBa_K2826001, BBa_K2826000, and BBa_k1357010.

In order to verify the copper adsorption efficiency of our copper treatment device (BBa_K2826013), two methods, including ICP in our lab and return the after-treated sample to RUNHE. As shown in figure 7, compared with control group, the copper concentration of test group treated with our device (BBa_K2826013) is obviously decreased. It proves that our device was working as we expected.

Figure 7: ICP results of copper ion concentration.

Figure 8: Curve of RFP fluorescence intensity and culture time under different copper concentrations of E.coli containing BBa_K2826013.

It can be confirmed by RFP fluorescence intensity that the expression of MbnABC is gradually enhanced as the concentration of copper ions increased (Figure 8). However, the results obtained by RUNHE is 6.11 mg/L, a little high than the initial concentration (Demonstration page for details). This may be due to excessive storage time and heating, etc., causing the water to volatilize.

To sum up, ICP results prove that our device was working as we expected, in other word, MbnABC gene can be expressed in E.coli to perform copper adsorption. However, there has not enough evidence to prove that our treatment device works normal in real samples, it needs to further optimized.

Future Work

We have seriously brainstormed about the methods we could use to control or regulate the copper ion capture ability of Mb. One method that has come to our minds is the use of the death gene. However, according to those calculated risks and our public survey, that we should not use those genes that could bring fear to the public. If available, the gene operon would be similar to our Detect 2.0, except we use death gene to replace the second chromoprotein, which will kill the bacteria when the copper level is lower than safe content.

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