iGEM 2019 — Team NWU-China

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# Team:NWU-China/Notebook

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# Notebook

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# Team:NWU-China/Experiments

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Experiment

1.Experiment of biosensor

1.1 Bacterial strains and growth conditions

    E. coli DH5α cells were used as the hosts for molecular cloning. They were cultivated in LB broth under shaking at 200 rpm at 37℃.

1.2 Measurements of fluorescence intensity

    Biosensor strains were grown in LB broth at 37℃ overnight. In a 96-well plate, The cells were diluted (1:50; v%) in 100μl of the M9 (supplemented with 0.4% glucose, 0.24 mg/mL MgSO4, 11.1μg/mL CaCl2, 0.3 μM thiamine hydrochloride, and 50μg/mL kanamycin) to each well, and Phe/Tyr was added at a concentration gradient of 0, 12.5, 25, 50, 100, 200μM. Place the 96-well plate into an automatic microplate reader. Incubate at 37℃ 24h and record the fluorometric value(530 nm/590 nm for RFP, 485 nm/528 nm for GFP) and OD600 for each well every 30 minutes.Each group should be repeated for at least 3 times.

![](https://static.igem.org/mediawiki/2019/1/1b/T--NWU-China--E_1.jpg)

1.3 Experiment of interference of urine components

    Since the experiment of collecting human urine involves ethical requirements, we decided to formulate it according to its main content in vitro.

|  |  |
| --- | --- |
| Main ingredient | Content |
| Water | 95% |
| Protein | 0% |
| Glucose | 0% |
| Urea | 1.8% |
| Uric acid | 0.05% |
| Inorganic salt | 1.1% |

Table 1: Main component of urine              \* Data From Wikipedia

    The components in the urine that affect the experimental results were mainly urea and uric acid, but it was unrealistic to culture cells with urine, so we configured a mother liquor of the M9 medium containing 1.8% urea and 0.05% uric acid. Because the Phe content in the urine of PKU patients was around 20mM (H.A. Harper et al., 1973), we added the engineered bacteria to the M9 medium (with 100μM Phe/Tyr) diluted 200 times urea and uric acid mother liquor, and set a blank control (no urea and urea added) and at 37℃, 200 rpm culture. The samples were taken at 8h, 12h, 16h, 20h, and 24h, and the fluorescence value and OD600 were measured in a microplate reader, and each experiment was repeated three times.

2. Experiment of colorimetric card

    Due to time issues, we only completed in vitro experiments in the color card section. We overexpressed the pigment proteins - amilCP, fwYellow, gfasPurple and spisPink in LB medium with E. coli, extracted and purified, and diluted to the same concentration. In a 96-well plate, the total volume of the pigment protein solution per well was 220μl, fwYellow was added according to a volume gradient of 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220μl, and then amilCP was added according to the opposite volume gradient. By the same token, in the same way, mix fwYellow and gfasPurple, spisPink and amilCP, spisPink and gfasPurple.

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# Team:NWU-China/Results

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Result

1.1 Experiment of Phe / Tyr

    We wanted to determine if there is a dose-response relationship between the Phe / Tyr concentration (concentration range 0-200μM) and the fold induction of the strain carrying PtyrP-rfp / ParoF-gfp. As shown in Fig.1 A, it could be clearly observed that the increase in Phe concentration had a significant effect on the activation of PtyrP and was linear (Fig.1 B, R² =0.9873). On the contrary, the increase in Tyr concentration had a significant repressive effect on ParoF (Fig.1 C) and had a strong linear relationship (Fig.1 D, R² = 0.9769).

![](https://static.igem.org/mediawiki/2019/f/ff/T--NWU-China--J_1.jpg)

1.2 Experiment of effects of urea and uric acid

    Because human urine contains 1.8% (0.29M) urea and 0.05% (2.9mM) uric acid, these may have an effect on bacterial growth and fluorescence induction, interfering with experimental results. We added a final concentration of 1.45 mM urea and 14.5μM uric acid (diluted two hundred times) in M9 medium containing 100μM Phe / Tyr, and measured OD600 and fluorescence intensity. The results show that urea and uric acid inhibit cell growth and fluorescence induction(Fig.2).

![](https://static.igem.org/mediawiki/2019/9/9e/T--NWU-China--Urea.jpg)

2. Experiment of colorimetric card

    We mixed the two pigment proteins (same concentration) in 96-well plates with a certain volume gradient. The mixed color was found to show a gradient change (Fig.3 A, B, C, D), and the gradient of the change produced by fwYellow and amilCP was most pronounced (Fig.3 A). To open, we added 100μl of fwYellow to each well, from 0 to 110μl, and added amilCP (Fig.3 G) to a volume gradient of 10μl, and added PBS to 210μl to each well. Instead, we added 100μlamilCP to each well, added yellow according to the volume gradient (Fig.3 H), and added PBS to 210 ul to each well. The results showed that the color change was significant and the difference was also visible to the naked eye.
    The colorimetric card provided a new approach to the future biosensor reporting system, in which a pigment protein was used as a often expression element and another pigment protein was used as a reporting system, depending on the induction intensity after a certain period of time. The resulting colors were also different. When there were two kinds of test substances, we could get the proportional relationship between the two substances according to the color change, but we couldn't know the content of the substances, which could be used as a semi-quantitative detection method.

![](https://static.igem.org/mediawiki/2019/b/ba/T--NWU-China--J_3.jpg)

Fig.3 Different pigmentprotein mixing results.
A. amilCP and fwYellow
B. gfasPurple and fwYellow
C. amilCP and spisPink
D. gfasPurple and spisPink
G. The concentration of fwYellow was fixed, and amilCP was added according to the volume gradient.
H. The concentration of amilCPwas fixed, and fwYellowwas added according to the volume gradient.

3. Conclusions

    In our project, we developed a biosensor for detecting Phe & Tyr in the urine of PKU patients and conducted initial experimental validation. We found that TyrR protein can produce different responses to Phe / Tyr concentration, and it had a strong linear relationship. Among them, Phe can activate PtyrP, while Tyr represses ParoF, thus expressing different fluorescence intensity, and detecting Phe in urine. The purpose of Tyr was to allow patients to understand their physical condition. However, the presence of urea and uric acid in the urine inhibits the growth of E. coli, which provided a reference for our biosensor post-correction data. The success of the colorimetric card pre-experiment provided a new idea for the future biosensor reporting system. The GFP/RFP reporting system required a specific instrument to view, which limited the use of the biosensor, so the visible color card system made the biosensor more accessible to the patient's home.

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