ERASynBIO:ECF Express - An Orthogonal, Organism-independent Expression Platform Based on Extractoplasmic Function (ECF) Sigma Factors

合成生物学的定义特征是既应用工程原理来推进生物学的理解，又为有用的目的开发新的功能。合成生物学的主要挑战之一是修改或创造比自然生物体更有效和可靠的新生物系统。本研究项目通过设计和实施新的调控模块来解决这样一个挑战，这些模块将控制和优化基因表达，而不会浪费细胞资源。这项研究的重大影响是教育下一代劳动力以支持不断增长的生物经济，为重要的生物过程提供新的基本理解，并为更广泛的合成生物学社区提供新的支持工具。本研究利用胞质外功能因子的合成生物学应用的新工艺。这些细胞质外功能因子（ECFs）经过重新设计后，可以作为强大的基因调控开关，能够控制多种转录活动。这些因子有两个保守的蛋白质结构域，可以识别和结合特定的启动子序列。然后它们招募rna聚合酶从天然或合成启动子位点启动转录。这些因子的组成简单性用于创建一系列针对特定调节需求量身定制的合成sigma因子/启动子对。在这个项目中，研究人员将从天然ECFs中创建嵌合ECFs，以生成一个大型的通用开关库，这些开关在四种模式细菌（大肠杆菌、S. meliloti、B. subtilis和S. venezuela）中是正交的。这将通过应用基于组合合成、结构引导突变方法和复杂遗传电路中的体内和硅测试的设计策略来完成。这一结果将为通过合成生物学实现生物设计提供新的工具。该项目是通过ERASynBIO欧盟-美国跨国资助机制资助的项目的美国合作部分。

The defining characteristics of synthetic biology are both the application of engineering principles to advance biological understanding and to develop new functions for useful purposes. One of the major challenges of synthetic biology is to modify or create new biological systems that are more efficient and reliable than natural organisms. This research project addresses one such challenge through the design and implementation of new regulatory modules that will control and optimize gene expression without wasting cellular resources. Significant broader impacts of this research are educating a next-generation workforce to support a growing bioeconomy, providing a new fundamental understanding of an important biological process, and generating new enabling tools for the wider synthetic biology community. This research takes advantage of a new process of using extracytoplasmic function sigma factors for synthetic biology applications. These extracytoplasmic function sigma factors (ECFs), when redesigned, can serve as robust gene regulatory switches capable of controlling diverse transcriptional activities. These factors have two conserved protein domains that recognize and bind to specific promoter sequences. They then recruit RNA-polymerase to initiate transcription from natural or synthetic promoter sites. The compositional simplicity of these factors are used for creating a series of synthetic sigma factor/promoter pairs tailored for specific regulatory needs. In this project, the investigators will create chimeric ECFs from natural ECFs to generate a large library of universal switches that are orthogonal in four model bacteria (E. coli, S. meliloti, B. subtilis and S. venezuelae). This will be accomplished by applying design strategies based on combinatorial synthesis, structure-guided mutational approaches and in vivo and in silico testing in complex genetic circuits. This results will provide new tools to enable biological design via synthetic biology. This project is the US collaborative component of a project funded through the ERASynBIO EU-US transnational funding mechanism.