使用合成性的生物迴路分析FimB與FimE的交互作用

Abstract

由於傳統實驗技術一直受到生物就有體內機制的限制及太多外來變因的影響。一個原因是因為正常生物體內的調控網路受到無數的調控因子調控，甚至有些調控因子尚未被科學家所發現。第二個原因是因為要分析生物體內交互作用及動態行為，在定量上會增加電腦計算的複雜度。為了能夠更精確地對內部的交互作用作定性及定量的分析，近年來許多生物學家從傳統生物轉向合成生物研究。藉由合成生物技術，科學家可突破生物體原有構造及功能的限制，使生物體可以產出科學家所希望產生的基因及蛋白質，進而去達到後續監測、治療、產能的目的。 許多革蘭氏陰性菌及革蘭氏陽性菌有線毛的構造，此結構能夠有利於菌體侵入宿主致病。線毛的產生與否，取決於fimS這段DNA片段是否轉向開啟下游線毛相關基因，fimS內具有啟動子，而FimB及FimE是兩個對fimS翻轉具有強大影響力的酪胺酸重組酶 FimE的催化是將fimS內的啟動子轉向不具有線毛基因的方向;而FimB具有雙向性，可關閉但傾向開啟線毛基因。兩酪胺酸重組酶均藉由結合在fimS兩旁的重複序列（IRL及IRR)，來決定線毛是否表達。 本篇研究，是建立一個合成生物系統， 藉由FimB及FimE對fimS序列的翻轉調控，來監測兩重組酶間隨著時間的互動性變化。建構方式主要利用國際基因工程競賽(iGEM)所提供的12個元件及2個重組酶FimB及FimE組成此系統。實驗結果發現，FimB與 FimE不只是隨著時間有競爭關係的現象，並且彼此之間的蛋白質產生速率差異，也會隨著誘導物濃度增加 ，而改變振幅及頻率。 可以清楚了解FimB、FimE兩競爭性蛋白質隨著時間變化在基因及蛋白質層面上的調控機制。此結果不但能驗證就有的在FimB及FimE的競爭概念，並能夠提供更詳細加入誘導物隨著時間的趨勢變化資料，有助於後續的定量分析。

To investigate dynamic behaviors in biological networks, many biologists have devoted to establish customized biological systems in vitro or in silico. The task of exactly presenting the interactive behavior is challengeable owing to two reasons: 1) the extra regulatory factors involved in the system with high interaction cannot be totally considered; and 2) the interactive and dynamic behaviors results in great computational complexity on quantitative analysis. This study aims to design a reversible and regulatory switch for investigating complex interactions in fim systems in vitro. The orientation of the promoter within fimS which is flanked by two internal repeats, IRR and IRL, is decided by the ratio of FimB and FimE site-specific recombinases. FimE mediates recombination dominantly in the ON-to-OFF direction which expresses fimA, however, FimB with a slight bias for OFF-to-ON direction. We incorporated 12 biobricks (from iGEM) and two recombinases, FimE and FimB into a new biological system where several interactive relations were included. We monitored red and green fluorescence proteins whose expressions affected by FimB and FimE. The results showed the interactive dynamic of FimE and FimB which is mutual competition over time with given concentrations of anhydrotetracyclin (aTc). And the difference of the expression rates affected by FimB and FimE is distinct at different concentrations of aTc.