Our detecting system is divided into two modules: The first module demonstrates regular gene expression to produce large amounts of b-galactosidase. A strong promoter is implemented in this module, so that E. coli can express downstream b-galactosidase functionally and stably. The second module is used for lysis, expressing lysis genes at later stages after detection of the target, via arabinose and copper-sensitive promoters. As the promoters detect the targets in its environment, E. Coli bursts open, and the previously produced b-galactosidase is released into the surrounding solution. After the addition of our chromogenic agent, x-gal, the solution will appear blue, so that the concentration of the detecting molecules could be measured by comparing greyscale values.
A normal functioning lysis module is crucial to our detecting system, as it allows exposure of b-galactosidase to the environmental solution. Although x-gal can diffuse through the membrane and induce a chromogenic reaction inside the cell, it would not be visible for us to observe without the lysis of cell wall. In other words, if it isn’t for the well-functioning lysis system, we would not be able to observe any visible color change.
Fig.1a Genetic circuit of lysis module
Our system is initiated by two components: pBad/araC promoter (BBa_I0500) and copper-sensitive promoter (BBa_I760005). Being two efficient and stable promoters, they induce the expression of lysis genes inserted in the bacterial plasmids productively, guaranteeing the working efficiency of our lysis module. Procedures to prove our lysis module using pBad/araC promoter:
1. Cultivate E. Coli in LB mediums at 37 degrees Celsius and 200 rpm.
2. When OD600 values equals to 0.3, add different concentrations of arabinose, three times for each group.
3. Measure OD600 values at 0.5h, 1h, 1.5h, 2h, 3h and 4h intervals.
Fig. 1b OD600 values of different concentration of arabinose at various timed intervals
The results suggested that the lysis circuit works regularly when the concentration of arabinose is above 10^-6 mol/L. The rapid decline of OD600 at 10^-5 mol/L indicates lysis of bacterial wall, which proves that our lysis module could function normally and continue to work in a relevant context.
Fig. 1c Comparison of OD values after 4h、10^-5 mol/L、10^-6 mol/L to control group (pBad/araC promoter)
Procedures to prove our lysis module using copper-sensitive promoter:
1. Cultivate E. Coli in LB mediums at 37 degrees Celsius and 200 rpm.
2. When OD600 values equals to 0.3, add different concentrations of arabinose, three times for each group.
3. Measure OD600 values at 0.5h, 1h, 1.5h, 2h, 3h and 4h intervals.
Fig. 1d OD600 values of different concentration of copper at various timed intervals.
Similar to the results from the pBad/araC promoter, the graph suggested that the lysis circuit works regularly when the concentration of copper is above 10^-6 mol/L.
Fig. 1e Comparison of final OD600 values after 4h、10^-6 mol/L、10^-5 mol/L (copper-sensitive promoter).
The chromogenic module is the key to the final presentation of our detection system's result. And the chromogenic module's results provide us with fundamental indicators for the qualitative and quantitative analysis of the subject. In our final product, the chromogenic module and the lysis module work together, but when verifying the chromogenic module alone, we use the engineered strain containing only the chromogenic module for verification. Furthermore, we used the arabinose promoter to induce β-galactoside's gene structures to avoid other factors influence.
The principle of the chromogenic module in our product is the chromogenic reaction between x-gal and endogenous bacterial β-galactosidase, which would produce the color blue. And the higher the concentration of the test substance, the greater the number of bacteria-induced to lyse will be. Consequently, there will also be more β-galactosidase participation in the chromogenic reaction, which causes results of color blue accumulation resulting in a darker gray value. Therefore, whether the chromogenic module presents the color blue as the result or other colors, it can still express the qualitative detection of the factor to be detected and whether it contains the element to be detected. On the other hand, if the module successfully displays the color blue, the gray value of the blue color can clearly show the density of the subject being tested. Furthermore, since x-gal can penetrate the bacterial cell membrane, it can react directly with its endogenous ß-galactosidase without lysis of the bacteria. Therefore, to ensure that all chromogenic reaction results are carried out by the ß-galactosidase released after the bacteria receive the signal to lysis, we will centrifuge the solution and take the supernatant for verification after waiting for the gene expression reaction.
The experimental steps of our verification experiment of the color rendering module are as follows:
1. Cultivate the engineered strain in the LB medium and wait until its OD600 value (surface bacterial concentration) reaches 0.4.
2. Add different concentrations of arabinose to the culture as a signal factor to initiate gene expression for more than half an hour.
3. Lyse the bacteria by sonication and take the supernatant after centrifugation for subsequent procedures.
4. Add x-gal and allow reaction for half an hour.
5. Observe the change of OD620 (absorbance value at 620 nm of the light wave) with a microplate reader.
Fig. 2a The OD620 value under different concentration of arabinose
As the fig. 2a shown, the OD620 value increases as the concentration of arabinose increases, which is due to the higher the arabinose concentration, the more ß- galactose is produced. And according to the previous argumentation in this passage, the gray value of the result will be higher. Consequently it will absorb more 620nm light wave, so OD620 value is higher. The result shows that the ß-galactose gene expressed properly and the relationship between the arabinose concentration and the OD620 meets the expectation.
Since the final production of our detection system will incorporate the function of the lysis and chromogenic modules working together, we performed a combined validation experiment after the two modules were independently validated. But since our verification experiment only includes verifying the effect of the lysis module and the chromogenic module, we will still use the arabinose promoter for the promoter that activates the two systems to prevent the promoter from affecting the experimental results. In our overall verification experiment, our arabinose promoter is the basics of the genes required for both modules to initiate.
The experimental steps we employed are as follows:
1. Cultivate the engineered strain in LB medium until its OD600 value (surface bacterial concentration) reaches 0.4.
2. Add different concentrations of arabinose to the culture as a signal factor to initiate gene expression, and at the same time, add a sufficient amount of x-gal, and incubate for about 3 hours.
3. Centrifuge the culture at 10,000 rpm for five minutes, and take the supernatant for subsequent testing.
4. Use a microplate reader to detect the OD620 value of the supernatant (absorbance value at 620 nm of the light wave)
Fig. 3a Comparison of final OD620 values / Comparision of OD620 10^-5 mol/L and control factor.
According to Fig. 3a, there is a positive correlation between concentration of arabinose and OD620 values. As shown in Fig. 3a, our system works best as a whole at arabinose concentration of 10^-5 mol/L, with the highest OD620 value, indicating highest optical density of the blue compound produced.
As b-galactocidase and x-gal undergo chromogenic reaction, a blue compound, varying in greyscale values, is produced. We measured its OD620 values, an indicator of optical density for our sample at a wavelength of 620 nm. By measuring OD620 values and hence the optical density of our sample, we could deduce the best working conditions for the system as a whole.
Fig. 4a Correlation between pH and OD620 values The optimal pH for our system to operate is around 6.8 to 6.9, which is very close to the neutral scale 7. Therefore, our product will not need supplementary solutions to adjust pH values. Since there is no requirement for an extreme acidic or alkaline condition, the modules are relatively easy to activate.
Fig. 4b Correlation between temperature and OD620 values OD620 is at its highest around 40 degrees Celsius, indicating our system's optimal temperature. Considering that our optimal temperature is higher than normal room temperatures, we've included heating wires in our testing kit to increase temperatures for the maximum working capacity.
In order to verify the accuracy and reliability of our detection system, we conducted various verification tests on the polluted areas in Dongdagou, Baiyin City, Gansu Province.
Baiyin City is an industrial city built for mineral development and was once known as the "Copper City." However, extensive mining and smelting have brought environmental hazards to the city. Especially before 1995, 22 industrial enterprises, including Baiyin Nonferrous Metals Company and Yinguang Company, discharged more than 19 million tons of metal-containing acid wastewater annually. The untreated industrial wastewater flows into Dongdagou and flows for 38 kilometers before entering rivers. As a result, the concentration of heavy metal pollutants exceeds the national discharge standard by 3 to 25 times, and a large number of heavy metals are deposited in the sediment of Dongdagou. At that time, data showed that cadmium pollutants within 100 cm depth of Dongdagou sediment exceeded the background value by 1400 to 2200 times, posing a severe threat to the water environment safety in the lower reaches of the Yellow River. Therefore, Dongdagou has become the largest sewage channel in Baiyin. Shui Qingchuan, vice director of Baiyin Institute of Environmental Science, said: "After decades of deposition, heavy metals such as copper, lead, zinc, arsenic, and other heavy metals exceeded the standards by a severe amount within 1 meter of the Dongdagou sediment. Among them, cadmium pollutants exceed the background value by 1400 to 2200 times, making Dongdagou the largest source of heavy metal pollution in the upper reaches of the Yellow River. In recent years, the Gansu provincial government has done a series of works on the heavy metal pollution in Dongdagou, which have led to substantial improvement.
In order to ensure the reliability of our verification experiment, we used a fully designed detection kit in this practical application. In addition, to ensure biosafety, all of our testing work is carried out in the laboratory after sampling in the field, ensuring that our engineered strains are not exposed outside the laboratory.
We selected many samples at different times for testing, including the Dongdagou copper-contaminated soil samples preserved before the Dongdagou treatment, the current surface soil samples of Dongdagou, and the current Dongdagou river water samples, current water samples 1 km downstream of Dongdagou Inlet River, current soil samples 1 meter deep in Dongdagou, current tap water samples in Baiyin City, current water samples from the Lanzhou section of the Yellow River, current soil samples in Baiyin City, and current lake water samples in Baiyin City.
Fig. 5a The results of different sampling samples in Dongdagou and other areas of Baiyin after being tested by our detection system are shown above.
As the Fig.5a demonstrates, the soil samples taken during the period of severe copper contamination ended up with a darker blue color, while the treated soil and water samples from Dongdaigou surroundings were both in a lighter blue. Samples taken 1m from the mouth of Dongdaigou into the Yellow River are further away from areas of severe contamination, but due to the contaminated water flows, the samples demonstrate a very light blue color. The other three samples taken in the uncontaminated center of Baiyin city were negative controls, none of which showed a blue color. In summary, all the results from our implementation prove experiments were running as expected, confirming that our detection system can work properly in a relevant context.
