Ninfa Alexander J, Selinsky Stephen, Perry Nicolas, Atkins Stephen, Xiu Song Qi, Mayo Avi, Arps David, Woolf Peter, Atkinson Mariette R
Department of Biological Chemistry, University of Michigan Medical School , Ann Arbor, Michigan, USA.
Methods Enzymol. 2007;422:488-512. doi: 10.1016/S0076-6879(06)22025-1.
Synthetic biology is an emerging field in which the procedures and methods of engineering are extended living organisms, with the long-term goal of producing novel cell types that aid human society. For example, engineered cell types may sense a particular environment and express gene products that serve as an indicator of that environment or affect a change in that environment. While we are still some way from producing cells with significant practical applications, the immediate goals of synthetic biology are to develop a quantitative understanding of genetic circuitry and its interactions with the environment and to develop modular genetic circuitry derived from standard, interoperable parts that can be introduced into cells and result in some desired input/output function. Using an engineering approach, the input/output function of each modular element is characterized independently, providing a toolkit of elements that can be linked in different ways to provide various circuit topologies. The principle of modularity, yet largely unproven for biological systems, suggests that modules will function appropriately based on their design characteristics when combined into larger synthetic genetic devices. This modularity concept is similar to that used to develop large computer programs, where independent software modules can be independently developed and later combined into the final program. This chapter begins by pointing out the potential usefulness of two-component signal transduction systems for synthetic biology applications and describes our use of the Escherichia coli NRI/NRII (NtrC/NtrB) two-component system for the construction of a synthetic genetic oscillator and toggle switch for E. coli. Procedures for conducting measurements of oscillatory behavior and toggle switch behavior of these synthetic genetic devices are described. It then presents a brief overview of device fabrication strategy and tactics and presents a useful vector system for the construction of synthetic genetic modules and positioning these modules onto the bacterial chromosome in defined locations.
合成生物学是一个新兴领域，在该领域中工程学的程序和方法被应用于改造活生物体，其长期目标是创造出有助于人类社会的新型细胞类型。例如，工程改造后的细胞类型可以感知特定环境，并表达作为该环境指标或影响该环境变化的基因产物。虽然我们距离生产出具有重大实际应用价值的细胞仍有一段路要走，但合成生物学的近期目标是对遗传电路及其与环境的相互作用形成定量理解，并开发出由标准的、可互操作的部件衍生而来的模块化遗传电路，这些部件可以引入细胞并产生一些期望的输入/输出功能。采用工程学方法，每个模块化元件的输入/输出功能被独立表征，从而提供了一个元件工具包，这些元件可以以不同方式连接以提供各种电路拓扑结构。模块化原理在很大程度上尚未在生物系统中得到验证，它表明当模块组合成更大的合成遗传装置时，它们将基于其设计特性正常发挥功能。这种模块化概念类似于用于开发大型计算机程序的概念，在大型计算机程序中，独立的软件模块可以独立开发，之后再组合成最终程序。本章首先指出双组分信号转导系统在合成生物学应用中的潜在用途，并描述了我们如何利用大肠杆菌的NRI/NRII（NtrC/NtrB）双组分系统构建用于大肠杆菌的合成遗传振荡器和拨动开关。描述了对这些合成遗传装置的振荡行为和拨动开关行为进行测量的程序。然后简要概述了装置制造策略和方法，并介绍了一种用于构建合成遗传模块并将这些模块定位到细菌染色体特定位置的有用载体系统。
