CIF: Medium: Collaborative Research: From Retroactivity to Modularity: Design and Implementation of a Genetic Insulation Device in Yeast

从追溯到模块化：酵母遗传隔离装置的设计和实现Domitilla Del Vecchio和罗恩Weiss模块化是工程系统的一个重要属性，但它是否是天然生物分子系统的一般属性是有争议的。发现模块性的程度并理解其机制是系统生物学中最重要的开放性研究问题之一。此外，合成生物学的长期成功关键取决于以这样一种方式实现模块化系统的能力，即单个组件的属性不会在它们相互连接时发生不可预测的变化。我们提出的研究旨在通过设计，实施和分析酵母中特殊的遗传追溯性绝缘装置，使用理论/实验相结合的努力来了解调控网络中的模块化机制。这种新的装置通过允许调节信号在正向方向上的传播同时最小化反向方向上的不期望的追溯现象，有效地使否则将高度互锁的转录组分重叠。除了为合成生物学家提供一个重要的新电路元件外，绝缘装置的数学分析将有助于更好地理解生物系统中调控网络的模块化程度。我们研究的第一个目的是表明，追溯性影响调控网络没有绝缘，因此，模块化不是生物分子信号通路的自然属性。其次，我们将展示一种新型的绝缘设备，该设备可以抵消追溯性，并允许电路在下游客户端加载的情况下可靠地传输信息。这种特殊的电路将被设计和放置在两个连接的组件之间，以使它们免受追溯效应的影响。我们要研究这个装置？的性能、调节下游组件的多个副本的能力以及正确操作的要求。第三，我们将研究绝缘设备与细胞环境的解耦程度。为此，我们将进行系统级分析，并研究设备和各种重要的细胞过程之间的串扰。 智力优势：现有的合成电路缺乏将驱动输入信号与输出负载的追溯性隔离的能力，从而排除了复杂生物电路的模块化组成。为了解决这个问题，我们建议构建和表征一种基于合成磷酸化的绝缘装置和仪器途径，这将展示一种用于构建复杂的大规模生物系统的通用和模块化技术。我们的新技术利用特殊的快速反馈机制集成到生物电路中，以创建绝缘设备，因此对许多其他生物图案和网络的设计和实现具有影响。我们的研究包括对具有反馈和追溯性的设备进行新的理论和计算分析，这些新工具也将适用于追溯性以外的生物网络问题的研究。这种分析对于调整和表征所需的绝缘性能，同时最大限度地减少对其他细胞过程的干扰至关重要。 更广泛的影响：在合成生物学中，这项研究将导致对生物分子系统设计的模块化工程原理的一般理解。同样，在系统生物学中，这项研究将解决一个基本问题？模块性在多大程度上是生物系统的固有属性？我们的合作成果将导致在自然系统中发现类似于我们的绝缘装置的图案，并解释如何实现模块化，包括洞察自然系统何时何地实现模块化以及用于什么目的。最终，模块化组合实现生物系统中定义的工程目标的能力将对人类治疗产生巨大影响，包括再生医学，糖尿病和癌症治疗，以及其他不同领域，包括生物燃料生产，环境修复，制药生产和生物传感应用。这项研究将有助于新的跨学科课程，并将被整合到一个为期十周的本科合成生物学暑期课程，最终在国际竞争中达到高潮，并有吸引妇女，代表性不足的群体和高中学生到该领域的跟踪记录。

From Retroactivity to Modularity:Design and Implementation of a Genetic Insulation Device in YeastDomitilla Del Vecchio and Ron WeissModularity is an important property of engineered systems, yet it is debatable whether it is a general property of natural bio-molecular systems. Discovering the extent of modularity and understanding its mechanisms is one of the most important open research problems in systems biology. Furthermore, the long term success of synthetic biology critically depends on the ability to implement modular systems in such a way that the properties of individual components do not change unpredictably upon their interconnection. Our proposed research seeks to understand the mechanisms of modularity in regulatory networks using a combined theoretical/experimental effort through the design, implementation, and analysis of a special genetic retroactivity insulation device in yeast. This novel device effectively decouples transcriptional components that would otherwise be highly interlocked by allowing propagation of a regulatory signal in the forward direction while minimizing the undesired phenomena of retroactivity in the reverse direction. Besides providing an important new circuit element for synthetic biologists, the mathematical analysis of an insulating device will lead to an improved understanding of the extent to which modularity is present in regulatory networks in biological systems. The first aim of our research is to show that retroactivity affects regulatory networks without insulation and therefore that modularity is not a natural property of bio-molecular signaling pathways. Second, we will demonstrate a novel insulation device that counteracts retroactivity and allows a circuit to transmit information reliably despite loading from downstream clients. This special circuit will be designed and placed between two connected components to insulate them from retroactivity effects. We will study the device?s performance, ability to regulate many copies of a downstream component, and requirements for correct operation. Third, we will study how well the insulation device is decoupled from the cellular environment. To this end, we will perform system-level analysis and investigate crosstalk between the device and various important cellular processes. Intellectual Merit: Existing synthetic circuits lack an ability to insulate a driving input signal from retroactivity of the output load, precluding modular composition of complex biocircuits. To address this problem, we propose to construct and characterize a synthetic phosphorylation-based insulation device and instrumentation pathway that will demonstrate a general and modular technique for building sophisticated, large scale biological systems. Our novel technique leverages the integration of special rapid feedback mechanisms into biocircuits in order to create insulation devices, and hence has implications for the design and implementation of many other biological motifs and networks. Our research includes new theoretical and computational analysis of devices with feedback and retroactivity, and these new tools will also be applicable for the study of biological network problems other than retroactivity. This analysis is fundamentally important for tuning and characterizing desired insulation properties while minimizing interference with other cellular processes. Broader Impact: In synthetic biology, this research will lead to a general understanding of the engineering principles of modularity for bio-molecular systems design. Likewise, in systems biology, this research will address a fundamental question ? To what extent is modularity an inherent property of biological systems? The product of our collaborative efforts will lead to the discovery in natural systems of motifs similar to our insulation device and to the explanation of how modularity is achieved, including insight into when and where natural systems implement modularity and for what purposes. Ultimately, the resulting capability of modular composition to achieve defined engineering goals in biological systems will have tremendous impact on human therapeutics, including regenerative medicine, diabetes, and cancer therapy, as well as in other diverse areas, including biofuel production, environmental remediation, pharmaceutical production, and biosensing applications. This research will contribute to new interdisciplinary courses and will be integrated into a ten week undergraduate synthetic biology Summer program that culminates in an international competition and has a track record of attracting women, under-represented groups, and high schools students to the field.