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

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.

This project aims to provide an expanded genetic toolkit for the construction, optimisation, and testing of synthetic cells. A requirement for the design of synthetic cells is the ability to modularly construct and edit DNA replicons, to precisely program selective gene expression and to manipulate multiple sets of genes that determine cellular structure and function. To build such programmable genetic circuits, orthogonally-acting genome editing tools that mediate predictable, controllable, and reversible rearrangements of DNA modules are required. Large serine integrases (LSIs) mediate precise and complete site-specific DNA recombination reactions, leaving not even a single nick in the DNA backbone. A simple choice of how the 40-50 bp DNA binding sites are arranged determines whether the reaction outcome is integration, excision, or inversion. Furthermore, LSI-catalysed reactions can be cleanly reversed by the addition of a second protein called a recombination directionality factor (RDF). These properties distinguish the LSIs from CRISPR-RNA-guided systems and from other site-specific recombinases. Synthetic cells are ideal candidates for genome editing with LSIs since the recombination sites can be built into predetermined loci in synthetic genomes. This project 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.