Team:NWU-China/Results
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).
Fig.1 (A) activation efficiency of PtyrP under different concentrations of Phe;
(C) repressive efficiency of ParoF under different concentrations of Tyr;
(B) and (D)Linear regression analysis of different concentrations of Phe / Tyr and fluorescence intensity(GFP/RFP) at 24th hours;
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).
Fig.2 Cell growth rate and fluorescence induction were measured by adding the main components of urine (1.48mM urea and 14.8μM uric acid) to 100μM of Phe (A & B) and Tyr (C & D).
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.
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.