<pclass="tab">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.</p>
<Pstyle="text-indent: 80px">Fig.1a Genetic circuit of lysis module</P>
<Pstyle="text-indent: 15px">Fig.1a Genetic circuit of lysis module</P>
<pclass="tab">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:</p>
<pstyle="text-indent: 80px">1. Cultivate E. Coli in LB mediums at 37 degrees Celsius and 200 rpm.</p>
<pstyle="text-indent: 80px">2. When OD600 values equals to 0.3, add different concentrations of arabinose, three times for each group.</p>
<pstyle="text-indent: 80px">3. Measure OD600 values at 0.5h, 1h, 1.5h, 2h, 3h and 4h intervals.</p>
<pstyle="text-indent: 15px">1. Cultivate E. Coli in LB mediums at 37 degrees Celsius and 200 rpm.</p>
<pstyle="text-indent: 15px">2. When OD600 values equals to 0.3, add different concentrations of arabinose, three times for each group.</p>
<pstyle="text-indent: 15px">3. Measure OD600 values at 0.5h, 1h, 1.5h, 2h, 3h and 4h intervals.</p>
<pstyle="text-indent: 80px">Fig. 1b OD600 values of different concentration of arabinose at various timed intervals</p>
<pstyle="text-indent: 15px">Fig. 1b OD600 values of different concentration of arabinose at various timed intervals</p>
<pclass="tab">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.</p>
<pstyle="text-indent: 80px">Fig. 1d OD600 values of different concentration of copper at various timed intervals.</p>
<pstyle="text-indent: 15px">Fig. 1d OD600 values of different concentration of copper at various timed intervals.</p>
<pclass="tab">Simialr 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.</p>
<pclass="tab">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.
</p>
<pclass="tab">The experimental steps of our verification experiment of the color rendering module are as follows: </p>
<pstyle="text-indent: 80px">1. Cultivate the engineered strain in the LB medium and wait until its OD600 value (surface bacterial concentration) reaches 0.4.
<pstyle="text-indent: 15px">1. Cultivate the engineered strain in the LB medium and wait until its OD600 value (surface bacterial concentration) reaches 0.4.
</p>
<pstyle="text-indent: 80px">2. Add different concentrations of arabinose to the culture as a signal factor to initiate gene expression for more than half an hour.
<pstyle="text-indent: 15px">2. Add different concentrations of arabinose to the culture as a signal factor to initiate gene expression for more than half an hour.
</p>
<pstyle="text-indent: 80px">3. Lyse the bacteria by sonication and take the supernatant after centrifugation for subsequent procedures.
<pstyle="text-indent: 15px">3. Lyse the bacteria by sonication and take the supernatant after centrifugation for subsequent procedures.
</p>
<pstyle="text-indent: 80px">4. Add x-gal and allow reaction for half an hour.
<pstyle="text-indent: 15px">4. Add x-gal and allow reaction for half an hour.
</p>
<pstyle="text-indent: 80px">5. Observe the change of OD620 (absorbance value at 620 nm of the light wave) with a microplate reader.
<pstyle="text-indent: 15px">5. Observe the change of OD620 (absorbance value at 620 nm of the light wave) with a microplate reader.
<pstyle="text-indent: 80px">Fig. 2a The OD620 value under different concentration of arabinose </p>
<pstyle="text-indent: 15px">Fig. 2a The OD620 value under different concentration of arabinose </p>
<p>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.</p>
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@@ -150,24 +150,24 @@ The result shows that the ß-galactose gene expressed properly and the relations
</p>
<pclass="tab">The experimental steps we employed are as follows:
</p>
<pstyle="text-indent: 80px">1. Cultivate the engineered strain in LB medium until its OD600 value (surface bacterial concentration) reaches 0.4.
<pstyle="text-indent: 15px">1. Cultivate the engineered strain in LB medium until its OD600 value (surface bacterial concentration) reaches 0.4.
</p>
<pstyle="text-indent: 80px">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.
<pstyle="text-indent: 15px">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.
</p>
<pstyle="text-indent: 80px">3. Centrifuge the culture at 10,000 rpm for five minutes, and take the supernatant for subsequent testing.
<pstyle="text-indent: 15px">3. Centrifuge the culture at 10,000 rpm for five minutes, and take the supernatant for subsequent testing.
</p>
<pstyle="text-indent: 80px">4. Use a microplate reader to detect the OD620 value of the supernatant (absorbance value at 620 nm of the light wave)
<pstyle="text-indent: 15px">4. Use a microplate reader to detect the OD620 value of the supernatant (absorbance value at 620 nm of the light wave)
<pstyle="text-indent: 80px">Fig. 3a Comparison of final OD620 values / Comparision of OD620 10^-5 mol/L and control factor. </p>
<pstyle="text-indent: 15px">Fig. 3a Comparison of final OD620 values / Comparision of OD620 10^-5 mol/L and control factor. </p>
<pclass="tab">According to Fig. 4d, 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.</p>
<h4><b>Optimal temperature and pH for our system as a whole</b></h4>
<pclass="tab">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.</p>
<pstyle="text-indent: 80px">Fig. 4a Correlation between pH and OD620 values
<pstyle="text-indent: 15px">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.</p>
<pstyle="text-indent: 80px">Fig. 4d Correlation between temperature and OD620 values
<pstyle="text-indent: 15px">Fig. 4d 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. </p>
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@@ -187,7 +187,7 @@ The result shows that the ß-galactose gene expressed properly and the relations
<pclass="tab">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.
<pstyle="text-indent: 80px">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. </p>
<pstyle="text-indent: 15px">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. </p>
