Team:Worldshaper-Shanghai/Results

Results

Part function test

As mentioned in DESIGN part, the ultimate goal of our project is to construct a portable, user-friendly blood test device which was designed based on a responsive regulatory system, named as HucR regulatory system. So first of all, the combination and dissociation of the HucR protein (BBa_K3311000) and pHucO operator (BBa_K3311001) need to be tested.

Part function test plan 1

As known in papers, the best way to verify the combination of HucR and pHucO is Electrophoretic Mobility Shift Assay (EMSA). But because of the limitation of the equipment of our lab, we decided to use an indirect way to verify it and designed two plasmids. At the same time, we continued to seek cooperation with other labs to do the EMSA experiment.

Assumption

In our first plasmid “pHucO-mCherry” (Figure 1), the mCherry will be promoted by pHucO. As for “HucR-pHucO-mCherry” (Figure 2), while uric acid is absent, HucR will bind to pHucO operator, inhibiting the transcription of downstream genes--mcherry. In this situation, the color of the sample will not change. While uric acid is present, it will change the structure of HucR, causing it to leave pHucO, allowing mCherry to transcript, the sample will turn red.

Figure 1 Schematic diagram of pHucO-mCherry

Figure 2 Schematic diagram of HucR-pHucO-mCherry

Results

The plasmid “pHucO-mCherry” was synthesised from company before use and it was transformed to E. Coli DH5α for function test. As shown in Figure 3, The bacteria turned red after incubating overnight at 37 °C, which means the pHucO works well.

Figure3. Incubation of E. Coli containing the pSB1C3-pHucO-mCherry

Then we successfully constructed the plasmid “HucR-pHucO-mCherry”（Figure4-5）. The plasmid contains three genes which is important for our part function test: HucR, pHucO, mCherry. HucR is promoted by a constitutive promoter, while the transcription of mCherry is controlled by pHucO.

Figure 4 Photos of transformants of pSB1C3-HucR-pHucO-mCherry on LB agar broth.

    

Figure 5 Enzyme digestion analysis pSB1C3-HucR-pHucO-mCherry.

The next step is induce the bacteria by using different concentration of uric acid. However, although the sample did turn light red when the bacteria were cultivated in solution containing uric acid, we did not observe any relationship between the concentration of uric acid and the intensity of fluorescence. Facing to the failure, we guessed that the problem might be related with the promoter.

Fortunately, we got very important suggestions from QHFZ-China 2019 iGEM team during an online meet up. The leader of their team told us that the pHucO we were using is too weak to let the gene pathway to produce enough mCherry. Plus, the promoter of HucR is too strong, causing a heaving repression on the transcription on mCherry. Furthermore, they provided two articles to support their suggestion1,2. According to their suggestion, we designed an improved plan: we added a strong constitutive promoter (BBa_J23100) in front of pHucO, enhancing the transcription of mCherry. In addition, the original strong promoter before HucR is replaced by a weaker constitutive promoter (BBa_J23110) so as to weaken the repression on pHucO, the pathway was registered as a composite part BBa_K3311005.

Figure 6 The new design of “HucR-pHucO-mCherry”

Reference：

1. Kemmer, C., Gitzinger, M., Daoud-El Baba, M., Djonov, V., Stelling, J., and Fussenegger, M. (2010) Self-sufficient control of urate homeostasis in mice by a synthetic circuit, Nature biotechnology 28, 355-360.
2. Wan, X., Volpetti, F., Petrova, E., French, C., Maerkl, S. J., and Wang, B. (2019) Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals, Nature chemical biology 15, 540-548.

Part function test plan 2

EMSA is a common affinity electrophoresis technique used to study protein–DNA or protein–RNA interactions, which is a direct way to prove the combination of HucR and pHucO. Many thanks for our team instuctors from Shanghai Cancer Institute, they helped us get in touch with the Herpesvirus and molecular virology research unit from Institute Pasteur of Shanghai & Chinese Academy of Sciences to do EMSA.

This experiment contains two items: biotin labeled pHucO and HucR-mCherry fusion protein. The biotin labeled pHucO was synthesized by company and the plasmid “HucR-mCherry-His” (Figure7) was constructed to express HucR-mCherry fusion protein.

The fusion protein purification is a necessary phase which is playing crucial role within the whole applied design test because our next experiment. We purified HucR-mCherry by His tag (Figure 8 and Figure 9), more detailed description of how the protein was purified was showed in protocols in our wiki.

Figure 7 Schematic diagram of HucR-mCherry.

Figure 8 Photos of the protein purification process.

Figure 9 The SDS-PAGE gel of purified protein.

(Notes: line1, whole bacteria; line2, supernatant; line3, flow-through; line4, 1st wash; line5, 2nd wash; line6, 1st elution; line7, 2nd elution; line8, 3rd elution)

The HucR-mCherry and biotin labeled pHucO were incubated together and then separated by EMSA gel. The HucR-mCherry-pHucO complex moves more slowly than non-binding labeled pHucO probes. After adding uric acid, HucR-mCherry-pHucO complex will decrease and could be viewed in EMSA gel. The concentration of HucR-mCherry increases from 0 to 1 g, which means the more HucR-mCherry is, the more the combination is, proving the combination of HucR-mCherry and pHucO (Figure10).

When uric acid appears in the solution, HucR-mCherry loses its function and falls off from the pHucO. Consequently, the downstream gene can be translated normally. The EMSA shows that, under the same concentration of HucR-mCherry and pHucO, there is a negative relationship between the concentration of uric acid and HucR-mCherry-pHucO complex. As the concentration of uric acid increases from 0 ~ 1000 M, the color of HucR-mCherry-pHucO complex become lighter (Figure11 ), indicating the effect of uric acid on HucR-mCherry-pHucO complex.

Figure 10 The combination of pHucO and HucR-mCherry in EMSA gel.

Figure 11. The combination of pHucO and HucR-mCherry after uric acid treatment.

Test paper test

The “cellulose acetate membrane” experiment

As one option concerning the applied design, the first thing to clarify is the mechanism behind the experiment. Two key elements (pHucO and HucR-mCherry) are utilized, the cellulose acetate membrane and the inverted fluorescence microscope. Another aspect which requires mentioning is about the in vitro environment. The pHucO DNA fragment is connected to the membrane solely, after that the fusion protein showing reddish simultaneously, will be added to the membrane, making the whole thing turn red. Assuring that the protein and the DNA fragment are perfectly binding together, then add different concentrations of uric acid into each membrane. Because various concentrations of uric acid can take off different amount of the fusion protein , the remains left on the membrane will be different (pHucO provides a binding site for the fusion protein, while the uric acid is added into it, the uric acid itself takes precedence over the HucR oppressor protein and changes its structure then the protein will get off from the binding site which pHucO provides). Eventually when the whole process is done, different sizes of red spots will be inspected on the membrane. Our team chooses the inverted fluorescence microscope to examine remaining spot. As it shown on Figure 12, with the increase of uric acid concentration, the number of red spots decreased either. For detailed analysis of the data, please check our wiki in Modelsection.

Figure 12 The red spot under different concentration of uric acid.

Future plan

To sum up, in this summer, we have achieved the product design, function verification of our main parts and the test paper product test. For us high school students, this experience is challenging but exciting. We want to do more in the future.

1. Optimize our test paper device, further improve the sensitivity and reproducibility of this device. Noticing the disadvantages in the way of fluorescence analysis by images, it is planned to establish an alternative Magnetic Beads Kit which is using the portable flourescence tester to collect the signals.
2. Advanced to the device itself, we would like to create an APP for phones that connects to the device through Wi-Fi or Bluetooth. Not just simply detecting the UA level, but also sending all the data to the APP. The APP will form a graph to present the changing of the UA level of the patient daily. Besides, it will also provide a personal diet plan based on the reported data in order to regulate the exceed UA level to a standard level.

Through our efforts, we wish it could bring convenience to patients who have gout and those who are likely to get gout in the way of saving their time to test UA level in hospitals. Those who have a high possibility to get gout because of their diet and habits can control their UA level at home before the situation getting more severe; in the long term, decreasing the potential patients of gout.

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