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应用工程原理来推进生物学理解，并为有用的目的开发新功能。合成生物学的主要挑战之一是修改或创建比自然生物更有效和可靠的新生物系统。该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正在教育下一代劳动力，以支持不断增长的生物经济，提供对重要生物过程的新的基本理解，并为更广泛的合成生物学社区产生新的使能工具。这项研究利用了一种新的方法，使用细胞质外功能西格玛因子的合成生物学应用。这些细胞质外功能σ因子（ECF），当重新设计时，可以作为强大的基因调控开关，能够控制不同的转录活性。这些因子具有两个保守的蛋白质结构域，其识别并结合特异性启动子序列。然后，他们招募RNA聚合酶从天然或合成的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监管需求定制的合成σ因子/启动子对。 在该项目中，研究人员将从天然ECF中创建嵌合ECF，以生成一个大型通用开关库，这些开关在四种模型细菌（E。coli、S.草木樨B. meliloti，B. subtilis和S.委内瑞拉）。这将通过应用基于组合合成的设计策略，结构指导的突变方法以及复杂遗传电路中的体内和计算机测试来实现。这一结果将为通过合成生物学进行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.