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，以方便询问有关基因组装如何共同发挥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各种创造性遗传操作（过去和未来）提供灵活和有用的工具，包括可编程遗传电路的模块化构建和方便编辑。这个工具包的核心是一个被称为“大丝氨酸整合酶（LSI）”的噬菌体酶家族，它介导精确的DNA重排。这些重排可以通过添加第二种称为重组方向性因子（RDF）的蛋白质来逆转。然而，RDFs很难在自然来源中识别，并且它们如何工作还不清楚。这项工作使用新的方法来解决这两个问题。该项目将显著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：一个大的面板的特点，正交LSI-RDF对;新的方法，为未来的研究人员来识别和/或工程LSI-RDF系统;结构信息和详细的理解需要推动进一步的工程，和新的光控功能。PI正在开发的方法，介绍高中学生看到和玩DNA的3D结构的兴奋，与社区团体合作，讨论现代遗传工具的潜在力量，并鼓励特别是妇女和代表性不足的群体注册大学STEM相关科目，并与生物技术行业合作，改进面向行业学生的课程。

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.