Left:
Lane pUC57-amilGFP: Plasmid pUC57-mini-vector-amilGFP digested by Bsa1 and the band at around 750bp
(amilGFP: 703bp) was got.
Lane pUC57-Pprovoin5: Plasmid pUC57-kana-mini-Pprovoin5 (2097bp) digested by Bsa1.
Right:
Lane pUC57-amilGFP: amilGFP (703bp) was got by PCR method.
Team:ECNUAS/Results
Results
IConstruction of recombinant plasmid
Figure 1: The electrophoresis results of enzyme digestion and PCR.
Figure 2: The electrophoresis (1) and sequencing (2) results of bacteria PCR.
Lane pUC57 -Pprovoin5-GFP-1 to 3: Bacteria PCR of monoclonals of amilGFP with size of 703bp. 1 to 3 were
positive monoclonals.
Extract 1 to 3 plasmids for sequencing.
Extract 1 to 3 plasmids for sequencing.
Figure 3: The bacteria C colony after transformation and selected by two antibiotics (kana&)
medium
Function tests
Table 1. Fluorescence intensity when the concentration of CYA equals to 30uM and the duration is 4
hours
Figure 4. Histogram of the fluorescence intensity when the concentration of CYA equals to 30uM and the
duration is 4 hours
As seen from figure 4, comparing to the blank control,
bacteria C presents an obvious higher fluorescence reaction to the cyanuric acid, the derivative from
Atrazine. In such a case, it could indicate that our engineered bacteria could work for detecting cyanuric
acid.
Table 2. Fluorescence intensity of bacteria C when when the duration is 4 hours under different
concentration of the cyanuric acid
Figure 5. Histogram of the fluorescence intensity of bacteria C when the duration is 4 hours under
different concentration of the cyanuric acid
Figure 6. Histogram of the fluorescence intensity of bacteria C when the duration is 6 hours under
different concentration of the cyanuric acid
In order to analyze the relationship between the concentration of cyanuric acid and
the fluorescence
intensity, we designed the control groups and collected the data as showing above. According to the
histograms (Fig. 5 and Fig. 6), the fluorescence intensity shows a decreasing trend with the increase of
concentration of cyanuric acid when we used the bacteria C for tests. Therefore, we speculate that the
cyanuric acid might affect the growth of strains so that the higher the concentration of the cyanuric acid,
the worse the growth of the bacteria, the less of the amount of the effective “biosensor”. In order to fully
eliminate this impact, we introduced the concept of cell-free extraction and cell-free expression in the
next stage of our project.
Figure 7. Curve of the fluorescence intensity of bacteria C against hours
In order to analyze the relationship between the fluorescence intensity and the
induction hours, we collected the data and drew the curves under various concentrations of cyanuric acid
(CYA) as showing above.
In figure 7, we can see that basically there is an increasing trend of the
fluorescence intensity of bacteria C as the induction hour increases. In addition, the curves also indicate
that the appropriate detection hour for our biosensor to detect cyanuric acid would be 4 hours later where
several curves tend to balance.
We followed the protocol and conducted the extract preparation for cell-free expression. In the end, we
obtained the extracted product, AtzR protein whose concentrations were measured by Nano 400 as shown in
table 3 with 1.5 ml each around.
Future Plan:
1. In order to further figure out the influence of cyanuric acid on the growth of bacteria C, there shall be
several control experiments to be conducted, such as to determine the growth curves of bacterial strains
under different concentrations of cyanuric acid.
2. As we just attempted the cell-free extraction once, this technique requires more experimental conditions groping to solid this part of work and collect more products for future cell-free expression use.
3. Once we obtain enough products of cell-free extraction, we could step into the cell-free expression experiments to ensure the performance of our biosensor without bacteria.
4. In order to achieve our final goal, the 3D print would be necessary for us to build the device which could load our cell-free biosensor and conduct more function tests with it. In the meantime, as the buffer in cell-free expression needs to be freshly configured, it is necessary to explore ways to preserve the product if it is made into a portable detection device.
5. In the future, we hope to carry our device to detect the content of cyanuric acid in lake/river water and other environmental water.
2. As we just attempted the cell-free extraction once, this technique requires more experimental conditions groping to solid this part of work and collect more products for future cell-free expression use.
3. Once we obtain enough products of cell-free extraction, we could step into the cell-free expression experiments to ensure the performance of our biosensor without bacteria.
4. In order to achieve our final goal, the 3D print would be necessary for us to build the device which could load our cell-free biosensor and conduct more function tests with it. In the meantime, as the buffer in cell-free expression needs to be freshly configured, it is necessary to explore ways to preserve the product if it is made into a portable detection device.
5. In the future, we hope to carry our device to detect the content of cyanuric acid in lake/river water and other environmental water.