21-BBSRC/NSF-BIO: Developing large serine integrases as tools for constructing and manipulating synthetic replicons.

这项合作工作开发了一个扩展的遗传工具包，以便于提出有关基因组合如何协同工作以执行自然或合成功能的基本问题。该工具包旨在为生物学家设想的(过去和未来)各种创造性的遗传操作提供灵活和有用的工具，包括可编程遗传电路的模块化结构和方便的编辑。这个工具箱的核心是一系列被称为“大丝氨酸整合酶(LSI)”的噬菌体酶，它们介导精确的DNA重排。这些重排可以通过添加另一种称为重组定向因子(RDF)的蛋白质来逆转。然而，RDF很难从自然来源中识别出来，它们的作用方式也不是很清楚。这项工作使用了新的方法来解决这两个问题。该项目将极大地扩展和创新可用于合成细胞研究的LSI-RDF工具包。我们的工作将提供：一个具有特征的、正交的LSI-RDF对的大小组；未来研究人员识别和/或设计LSI-RDF系统的新方法；推动进一步工程所需的结构信息和详细了解，以及新的光控制功能。PI正在开发方法，使高中生对观看和玩DNA的3D结构感到兴奋，与社区团体合作，讨论现代基因工具的潜在力量，并鼓励大学STEM相关课程的注册，特别是在女性和代表性不足的群体中，并与生物技术行业接触，以改进行业学生的课程。

This collaborative work develops an expanded genetic toolkit to facilitate asking fundamental questions about how assemblies of genes work together to perform natural or synthetic functions. This toolkit is intended to be flexible and useful for the broad variety of creative genetic manipulations envisioned (past and future) by biologists, including the modular construction and convenient editing of programmable genetic circuits. The core of this toolkit is a family of bacteriophage enzymes termed "Large serine integrases (LSIs)" that mediate precise DNA rearrangements. These rearrangements can be reversed by the addition of a second protein called a recombination directionality factor (RDF). However, RDFs have been difficult to identify in natural sources, and how they work is not well understood. This work uses new approaches to address both of those problems. This project will significantly expand and innovate the LSI-RDF toolkit that is available for use in synthetic cell studies. Our work will provide: a large panel of characterized, orthogonal LSI-RDF pairs; new approaches for future researchers to identify and/or engineer LSI-RDF systems; structural information and detailed understanding needed to drive further engineering, and new light-controlled functionality.The PIs are developing ways to introduce high school students to the excitement of seeing and playing with the 3D structures of DNA, working with community groups to discuss the potential power of modern genetic tools and to encourage enrolment to university STEM-related subjects especially among women and underrepresented groups, and interfacing with the biotechnology industry to improve curriculum for industry-bound students.