UKRI/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，以促进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。该工具包的核心是一个称为“大丝氨酸整合酶（LSI）”的酶家族，其介导精确的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。 PI正在开发方法，让高中生看到和玩DNA的3D结构，与社区团体合作，讨论现代遗传工具的潜在力量，并鼓励大学STEM相关学科的入学，特别是在女性和代表性不足的群体中，并与生物技术产业对接，以改善产业课程-来自芝加哥大学的美国研究员Rice和来自利物浦约翰摩尔大学的英国研究员Olorunniji的这项合作研究汇集了两个在丝氨酸重组酶方面具有长期专业知识的团队。 Olorunniji集团在合成生物学和分子生物学方面的专业知识与Rice集团在结构生物学方面的专业知识相辅相成。 他们的共同努力将显着扩展和创新可用于合成细胞研究的LSI-RDF工具包。 该项目将：（1）使用新的和已建立的计算方法鉴定具有有用性质的新型整合酶- RDF对，随后在体内功能测定中进行表征;（2）使用结构生物学的工具来理解RDF如何与它们的同源LSI相互作用，为蛋白质工程提供坚实的基础;（3）开发新的方法来缓解当前识别RDF的瓶颈，以及（4）通过将RDF设计为光响应，为工具箱添加新的功能，以快速识别RDF。不需要化学诱导剂的反应反应逆转。这一美国/英国合作项目由美国国家科学基金会（NSF）和英国生物技术和生物科学研究理事会（BBSRC）支持，其中NSF资助美国调查员，BBSRC资助英国的合作伙伴。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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 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. 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.This collaborative study between US investigator Rice from the University of Chicago and UK investigator Olorunniji from Liverpool John Moores University brings together two groups with long-standing expertise in serine recombinases. The Olorunniji group’s expertise in synthetic biology and molecular biology is complemented by the Rice group’s expertise in structural biology. Their combined efforts will significantly expand and innovate the LSI-RDF toolkit that is available for use in synthetic cell studies. The project will: (1) identify novel integrase - RDF pairs with useful properties using both new and established computational approaches, followed by characterization in in vivo functional assays; (2) use the tools of structural biology to understand how RDFs interacts with their cognate LSIs, providing a solid foundation for protein engineering; (3) develop novel methods to alleviate the current bottleneck in identifying RDFs and (4) add new functionality to the toolbox by engineering RDFs to be light-responsive for rapid-response reaction reversal without the need for chemical inducers.This collaborative US/UK project is supported by the US National Science Foundation (NSF) and the UK Biotechnology and Biological Sciences Research Council (BBSRC), where NSF funds the US investigator and BBSRC funds the partners in the UK.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.