利用可控制生物元件及回饋設計增加基因迴路之穩定性

Abstract

合成生物學意指利用不同生物元件組合成基因迴路，讓其依循可預期的方式表現出特定功 能。計畫中將建構不同轉錄強度的啟動子，並在啟動子上組合抑制子及活化子結合序列，增加 生物元件的可選擇性及可調控性，建立多樣化的生物元件資料庫。將這些生物元件構築於質體 中，便能組成基因迴路，控制宿主細胞進行一系列的工作。但是基因迴路在細胞中會遭受外界 環境及宿主細胞內部訊息干擾，使外加的基因迴路無法穩定運作。為解決此問題，我們將建立 數學模型描述基因迴路動態行為，將回饋機制設計於基因迴路中，增加迴路輸出訊號之穩定性， 降低不同細胞間基因表現量的差異。之後利用實驗數據修正基因迴路運作的動態方程組，使基 因迴路的表現可以由數學模型模擬再現，進一步預測出不同基因迴路運作的動態形式。 本計畫整合模擬及實驗，可定性及定量描述各生物元件及基因間的交互作用，由下而上更 瞭解基因網路間的調控方式，提出更佳方式來建構穩定的生物基因迴路。而且當生物元件資料 庫擴增後，可以依各元件特性，用電子電路設計概念，由生物元件資料庫中組合出最適合的基 因迴路，用來改造菌株。這項計畫的成果將可應用於設計經濟效益最佳的能源生產途徑，並拓 展到產業應用。

Synthetic biology is the design and construction of new biological parts, devices, and systems, and the re-design of existing, natural biological systems for useful purposes. Genetic engineering with recombinant DNA is a powerful and widespread technology that enables researchers to redesign life forms by modifying DNA fragments. However, most created genetic circuits are unstable and can not function properly. Therefore, how to design a stable genetic circuit to tolerate intrinsic parameter fluctuation and to attenuate extrinsic disturbances in order to function properly is an important topic for synthetic biology. In this project, we will use controllable biobricks and the feedback loop concept to increase stability of gene circuits to meet this challenge. The first step in programming and controlling cell behavior is to establish a library of well-defined components—‘biobricks’ that serve as the building blocks of artificial gene networks. Biobricks are the DNA fragments with specific functions, including promoters, repressors, reporter genes and other parts required to construct functional plasmids. The main challenge in genetic circuit design lies in selecting well-matched genetic components that when coupled, reliably produce the desired behavior. Although the parameter values are calculated by model equations, it is hard to select the biobricks that reliably implements a desired cellular function with quantitative values. To overcome this problem, the promoters which can control the expression of downstream genes are necessary. Two strategies will be applied. First, degenerated primers designed for PCR are used to generate mutations in promoter regions. Second, the combinatorial promoter library with multiple transcription factor binding sites facilitating the integration of multiple signals will be constructed. The promoter activity can be assay using a bacterial luciferase reporter cassette on a low copy number plasmid. A library of promoters with different transcriptional strength will be built to tune the specific parameter values that model equations indicated. After building genetic circuit, the experimental data can provide the mathematical property of parameters and refine the circuit design schemes to increase stability of systems. The stable and noise-resistance circuit design schemes have potential for applications, such as constructing a synthetic enzymatic pathway for drug production, and producing metabolic pathway in microbes that churn out bio-fuels.