開發以轉錄因子為基礎的全細胞生物感測器用於檢測苯丙胺酸與酪胺酸

Development of Transcriptional Factor-Based Whole-Cell Biosensors for Detection of Phenylalanine and Tyrosine

林潔(Lin Chieh)

指導教授 ： 葉怡均

國立臺灣師範大學/理學院/化學系/碩士(2018年)

https://doi.org/10.6345/THE.NTNU.DC.025.2018.B05

摘要

芳香族胺基酸在生物體內有許多重要的功能，是神經傳導物質、生長激素或是賀爾蒙的前驅物，在生物體內的濃度維持恆定，若過多過少都會造成疾病，例如苯丙酮尿症、酪胺酸血症等。人體內的酪胺酸大多是由苯丙胺酸轉變而來，這兩者之間的濃度也會互相影響。本實驗針對苯丙胺酸及酪胺酸設計了兩種全細胞生物感測器，第一部分利用TyrR調控蛋白控制芳香族胺基酸合成的機制，TyrR會對其調控組有不同程度的調控，研究各基因啟動子在苯丙胺酸或酪胺酸環境下的表現，選擇ParoF與PtyrP作為感測器的設計，並以螢光蛋白作為訊號表現，兩個啟動子分別對酪胺酸及苯丙胺酸都有良好的線性範圍與偵測極限。第二部分對PtyrP啟動子作突變，針對酪胺酸做感測，以兩種螢光蛋白作為訊號，可以依據酪胺酸濃度高低的不同，而呈現不同顏色的螢光訊號，兩種訊號都有良好的線性範圍與偵測極限。胺基酸代謝異常除了在血液中有顯著的濃度變化之外，也會影響其在尿液中的含量，尿液的收集是一種非侵入性的方式，本實驗最後也有將兩個部分所設計的感測器，嘗試用來檢測尿液中苯丙胺酸與酪胺酸的含量，並與液相層析質譜儀 (LC-MS) 的數值進行比較。

關鍵字

苯丙胺酸 ； 酪胺酸 ； 轉錄因子TyrR ； 全細胞生物感測器

並列摘要

Aromatic amino acids play important roles in many organisms. They are the precursors of neurotransmitter, auxin and hormones. The levels of amino acids are normally maintained in a certain range, and out of range may lead to diseases, such as phenylketonuria and tyrosinemia. Tyrosine in human is mostly converted from phenylalanine, and the concentrations of the two can also affect each other. In this study, we developed two whole-cell biosensors for phenylalanine and tyrosine. In the part I, we utilized the transcriptional regulator TyrR to investigate the gene expressions of TyrR regulon promoters in the presence of phenylalanine or tyrosine. TyrR is the protein which regulates the synthesis and transport of aromatic amino acids by activating or repressing the genes of TyrR regulon. We selected PtyrP and ParoF promoters for our biosensor design. The two promoters both had good specificity, linear range and limit of detection for phenylalanine and tyrosine, respectively. In the part II, we mutated the PtyrP promoter for sensing tyrosine only. The sensor could respond to the tyrosine levels with two color fluorescent proteins. When the tyrosine level was low, the sensor presented red fluorescence; when the level was high, the sensor presented green fluorescence. The two signals had good linear range and limit of detection. Finally, we also test the sensors in human urine. The abnormal amino acid metabolism is not only reflected in blood but also in urine, and urine collection is a non-invasive way. We used the sensors to determine phenylalanine and tyrosine levels in human urine and compared with liquid chromatography-mass spectrometry (LC-MS).

並列關鍵字

phenylalanine ； tyrosine ； transcriptional regulator TyrR ； whole-cell biosensor

參考文獻

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