Team:NWU-China/Design
Project Design
1. Introduction
We want to design a new type of biological test strip that, when reacted with human urine, exhibits a specific color, and then compares it to our own designed colorimetric card to determine phenylalanine / tyrosine in human urine. Our ideal system is specific and sensitive to phenylalanine and tyrosine without interference from other amino acids. Therefore, our entire experiment is divided into two parts: building a biosensor and designing a color chart.
2. Biological sensor
Phenylketonuria (PKU) causes an increase in Phe levels due to a deficiency in phenylalanine hydroxylase (PAH). PAH plays an important role in the hydroxylation of phenylalanine (Phe) to tyrosine (Tyr). Besides, an increase in the Phe / Tyr ratio is usually observed in PKU patients. This suggests that continuous measurement and quantification of two Phe and Tyr may be helpful to maintain Phe that is strictly controlled in dietary intake, thereby preventing emotional instability or physical dysfunction. Besides, the phenylalanine / tyrosine ratio has been used to confirm the effective diagnosis of PKU and hyperphenylalaninemia.
The biosynthesis of Phe and Tyr in E. coli is controlled by the DNA binding protein TyrR. The TyrR protein binds to the consensus DNA sequence TGTAAAN6TTTACA (referred to as the TyrR box), which is classified as strong or weak. In the presence of Phe, binding of the TyrR dimer to the strong cassette in vitro strongly induces gene expression by promoting binding of the RNA polymerase to the promoter or promoting binding of the second dimer to the adjacent weak box. In the presence of Tyr and ATP, the TyrR dimer self-associates to form a hexamer, and the hexamer binds weakly to the weak consensus of the consensus sequence, due to weak-frame-bound RNA polymerase forming an open complex or leaving the initiation impaired ability of the child leads to inhibition of gene expression. Thereby regulating the repression of aroF, tyrP. (Pittard J et al., 2005)
3. Phe/Tyr test device
Based on the literature and experimental principles, we constructed a detection pathway. We hope to determine the Phe/Tyr content according to the expression of amilcp / fwyellow, thus reflecting the physical condition of patients with phenylketonuria and helping them to change their living habits.
4. Colorimetric card
In the initial experiment, we quantified the ratio of Phe / Tyr by the intensity of expression of fluorescent protein. We considered that if biological test strips are used in daily life, the intensity of fluorescent protein expression is difficult to observe and measure, and the pigment protein is easy to observe with the naked eye, so we want to replace the fluorescent protein with pigment protein. Moreover, we know that two different color pigments will be mixed at different concentrations to get different colors. For example, different concentrations of yellow and blue will give different colors of green, so will pigment proteins have such properties? Therefore, we selected yellow pigment protein (fwyellow, K1033910), blue pigment protein (amilCP, K592009), pink pigment protein (spisPink, K1033932), purple pigment protein (gfasPurple, K1033919), which were mixed according to a series of gradient concentrations. We hope to get a series of color gradients and get a standard color chart to provide a simple, portable reporting system for future biosensors.