用系統工程方法設計強健生物IC電路---子計畫三：以定向演化達成合成途徑最佳化與驗證強健系統設計理論

Abstract

合成生物學是現代生物學的新興研究領域，其利用基因重組技術製造各種生物元件，組合 成生物IC電路，讓其依循可預期的方式表現出特定功能，控制細胞進行一系列的工作。現今架 構生物IC電路面臨的難題是生物電路在細胞內表現短暫功能後會逐漸崩潰，無法穩定運作於細 胞中。基因表達時的雜訊，不同RNA剪裁、DNA突變及熱擾動等造成內在生化參數變動，以 及細胞環境變遷與其他基因交互作用所造成的干擾，都會使人工製造出來的基因電路無法穩定 運作。因此如何設計可以忍受內在及外界干擾的強健基因網路，是生物IC電路亟待解決的重要 課題。 在生物體中建構基因電路需要瞭解生物細胞中許多未知的生化參數，例如mRNA及蛋白質 的穩定性，DNA與蛋白質的交互作用等等。為了克服此難題，我們將建立生物元件DNA 序列 資料庫並量測其輸出訊號表現強度，此資料庫可以提供構築基因電路所需之生物元件。不過現 今生物元件庫組合還不夠多，往往只能先組裝出最接近設計藍圖的生物IC電路。這種無法精確 組裝的生物IC電路不見得可以在宿主細胞中穩定運作，這也是現今已發表的生物電路無法穩定 表現的原因之一。 本子計畫將引入定向演化技術改良生物IC電路組裝無法逼近設計藍圖的問題。利用定向演 化使啟動子轉錄強度發生變化，我們再由不同調控強度組合的生物IC電路中，選取最接近設計 藍圖的子代。實驗過程將回傳生物電路輸入及輸出訊號給一到五的合作子計畫，分析達成生物 IC電路強健化重要因素。比對模擬與實驗結果，修正強健生物IC理論，再將新的生物電路設 計圖回傳給本子計畫驗證強健系統設計理論。依據設計理論架構，我們將於大腸桿菌中組裝最 佳化生質丁醇量產途徑，使生產丁醇的經濟效益提昇到有工業應用價值。這項整合性計畫的成 果將可利用於設計經濟效益最佳的能源及化學產物生產途徑，應用於未來生質能源領域。

The main goal of the nascent field in synthetic biology is to design and construct biological system with a desired behavior. At present, even the construction of biological IC circuits has demonstrated the feasibility of synthetic biology, the design of gene networks is still a difficult problem and most of newly designed gene networks can not function properly. The major causes for these design failures are mainly due to both intrinsic perturbations such as gene expression noise, splicing, mutation, evolution and extrinsic disturbances such as changing extra-cellular environments, interactions with cellular context. Therefore, how to design a robust synthetic gene network to tolerate intrinsic parameter fluctuation and to attenuate extrinsic disturbances in order to function properly will be an important topic for synthetic biology. However, building a genetic circuit in vivo requires tedious measurement of many unknown parameters of mRNA, protein stabilities, DNA-protein interactions and so on. To overcome this problem, we will generate a library of genetic devices with a range of behaviors that can be used to construct genetic circuit and develop evolutionary strategies for constructing biological IC circuits. A combined rational and evolutionary design strategy are proposed for constructing biological IC circuits, an approach that allow the engineer to fine-tune the biochemical parameters of the gene regulatory networks experimentally in vivo. By applying directed evolution to genes comprising a genetic circuit, the non-robust genetic circuit containing improperly matched components can evolve rapidly into a more robust one. The experimental data can provide the mathematical properties of parameters and our team can refine the circuit design schemes to increase robustness of bio-systems. In next step, we will use the robust genetic circuit design to engineer an Escherichia coli strains to product butanol from glucose via the host’s amino acid biosynthetic pathway. Design of genetic circuits is foreseen to have important applications in biotechnology, medicine and biofuel, and to revolutionize how we conceptualize and approach the engineering of biological systems.