标题: | 以模型设计方法建构可预测功能之基因电路 Construction of genetic circuit with predicted functions by model-based design method |
作者: | 高敏智 Kao, Min-Chih 曾庆平 Tseng, Ching-Ping 生物科技学系 |
关键字: | 合成生物学;基因回圈;生物砖;模组;synthetic biology;genetic circuit;biobrick;model |
公开日期: | 2011 |
摘要: | 合成生物学是现代生物学的新兴研究领域,其利用基因重组技术制造各种生物元件,组合成生物基因回路,让其依循可预期的方式表现出特定功能,控制细胞进行一系列的工作。DNA可视为构筑生物系统的蓝图,DNA转译出来的蛋白质则扮演着执行者的角色。藉着不同蛋白质间的交互作用,可以让生物稳健的生存并表现出特殊功能。蛋白质的表现量主要受控于启动子(promoter)负责调控的转录层级以及核糖体结合区(ribosomal binding site)控制的转译层级。本篇论文中,引进了iGEM元件系统进行DNA设计与组装。我们将启动子以及核糖体结合区整合为一个控制蛋白质表现量之功能装置,并定量蛋白质表现量与细菌密度之关系。当下游基因搭配不同启动子与核糖体结合区域,即会有相对应的蛋白质表现量输出。当设计者预期表现特定量的蛋白质,只需从我们建立的资料库中挑选相对应组合的蛋白质表现之功能性装置后,即可得到预期的蛋白质表现量。 在实验设计上,我们在分成三部份进行: 1) 建构蛋白质表现系统质体并测量其蛋白质输出:我们从iGEM元件库中挑选了四个不同转录强度的启动子搭配三个不同转译强度的核糖体结合区,总共十二种组合的蛋白质表现之功能性装置。以绿萤光蛋白当下游基因进行表现,并用流式细胞仪侦测其随着时间的萤光讯号输出。 2)建立数学模型定量每组蛋白质表现之功能性装置:藉由第一部分的实验数据进行蛋白质表现系统数学模型的建立并定量蛋白质表现系统表现强度与细菌密度之关系。 3)系统应用及验证:我们设计表现抑制蛋白控制下游基因表现之基因回路,系统中具有上游基因表现抑制下游基因表现输出的行为。两个系统皆可以利用第二部分参数预测其交互作用后的行为与蛋白质输出量,证实元件规格化理论可用在复杂基因回圈中。 本篇论文中整合模拟及实验,可定性及定量描述各生物元件及基因间的交互作用,由下而上更瞭解基因网路间的调控方式,提出更佳方式在大肠杆菌中建构稳定的生物基因回路。而且当生物元件资料库扩增后,可以依各元件特性,用电子电路设计概念,由生物元件资料库中组合出最适合的基因回路,用来改造菌株。这项计画的成果将可应用于设计经济效益最佳的能源生产途径,并拓展到产业应用。 Genetic engineering with recombinant DNA is a powerful and widespread technology that enables researchers to redesign life forms by modifying DNA fragments. Programming and controlling cell behavior requires fine control the protein expression levels. Previous studies provide several methods to predict the transcription rates of promoters and translation rates of ribosome binding sites (RBSs) respectively. However, the protein expression level with time is hard to predict properly by those methods. To overcome this problem, we selected four promoters and three RBSs with different regulation strength and constructed 12 protein expression devices which combine promoter, RBS and green fluorescent protein (GFP) in Escherichia coli. The GFP expression levels with time were measured using a flow cytometry, and the experimental data can used to characterize a protein expression rate of a protein expression device which contains a promoters and a RBS. A dynamic model that captured the experimentally observed differences for each protein expression device was developed in this study. Using this model, we can define the protein expression rate in the different E. coli population density for the protein expression device. To demonstrate reverse engineering, this model was used to predict the protein expression level in repressor-controlled genetic circuits, and the experimental results consistent with our prediction. Thus, this model enable us to rational connect a promoter and a RBS to obtain a target protein expression level in a complex genetic circuits. Our method can quantitative the protein expression rate at different E.coli population density and can implement a genetic circuit with desired function in E.coli. |
URI: | http://140.113.39.130/cdrfb3/record/nctu/#GT079828512 http://hdl.handle.net/11536/47720 |
显示于类别: | Thesis |