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Engineering of artificial cellular circuits based on the LuxI-LuxR quorum-sensing system.

机译：基于LuxI-LuxR群体感应系统的人工蜂窝电路工程。

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摘要

Natural cellular networks are very good at processing diverse inputs, generating complicated responses, and confounding researchers with their complexities. As an alternative to traditional cellular engineering approaches, the field of synthetic biology attempts to avoid the complexities of natural systems by focusing on the bottom-up construction of artificial cellular circuits. By rationally building up circuit complexity, synthetic biologists hope to both create novel systems capable of programming unique cellular responses, and gain insights into the design principles of natural systems. Circuits that allow for the programming of intercellular responses are of particular interest, and researchers have focused on the use of bacterial communication mechanisms (quorum sensing) to construct such circuits. At their most basic, quorum-sensing systems are composed of three main components, making them amenable to genetic manipulation. These components, however, have properties that have been finely tuned through evolution to function in very specific ways, and repurposing them for our own uses requires methods to overcome their naturally evolved properties.;This thesis details our work in the construction and engineering of synthetic circuits based on components of the LuxI-LuxR quorum-sensing system. Using these components, we demonstrate methods for altering both the sensitivity and the form of the quorum-sensing response through the creation of three unique systems: an ultrasensitive positive feedback loop, a logical AND gate, and a coupled feedback loop oscillator. Construction and tuning of each circuit's properties were achieved through a mixture of rational and evolutionary approaches, with particular emphasis on the directed evolution of the LuxR transcriptional activator. Mathematical modeling was also used during the construction of the more complex circuits to predict the properties that were essential to their functionalities. With the construction and characterization of these circuits, we have provided both well-defined modules that can be used in the construction of more complex systems, and developed methods that will allow for the creation and engineering of additional synthetic circuits.
机译：天然蜂窝网络非常擅长处理各种输入，生成复杂的响应以及使研究人员感到困惑的复杂性。作为传统细胞工程方法的替代方法，合成生物学领域试图通过关注人工细胞电路的自下而上构造来避免自然系统的复杂性。通过合理地建立电路复杂性，合成生物学家希望既能创建能够对独特细胞反应进行编程的新颖系统，又能洞悉自然系统的设计原理。允许对细胞间反应进行编程的电路特别受关注，研究人员已将精力集中在利用细菌传播机制（群体感应）构建此类电路上。在最基本的群体感应系统中，它由三个主要组成部分组成，使其易于进行基因操作。但是，这些组件的特性已经通过演化以非常特定的方式进行了微调，因此，将它们重新用于我们自己的用途需要克服其自然演化的特性的方法。；本文详细介绍了我们在合成材料的构建和工程中的工作电路基于LuxI-LuxR群体感应系统的组件。使用这些组件，我们演示了通过创建三个独特的系统来改变灵敏度和群体感应响应形式的方法：一个超灵敏的正反馈环路，一个逻辑“与”门和一个耦合反馈环路振荡器。通过合理和进化方法的混合来构建和调节每个电路的特性，尤其着重于LuxR转录激活因子的定向进化。在构建更复杂的电路时，还使用数学建模来预测对其功能必不可少的特性。通过这些电路的构造和特性，我们既提供了可用于构建更复杂系统的定义明确的模块，又提供了可用于创建和设计其他合成电路的开发方法。

著录项

  • 作者

    Sayut, Daniel J.;

  • 作者单位

    University of Massachusetts Amherst.;

  • 授予单位 University of Massachusetts Amherst.;
  • 学科 Engineering Chemical.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 161 p.
  • 总页数 161
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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