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

University Of Chicago, Chicago IL

Investigators

Abstract

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.