SUMMARY- CORE 1: EXPRESSION AND MOLECULAR BIOLOGY (EMB) CORE High quality protein samples are rate limiting for most structural and mechanistic investigations of DNA Repair (DR) complexes and assemblies. Core 1, the Expression and Molecular Biology (EMB) Core, is designed to lower these barriers for individual labs by providing access to centralized resources and expertise in advanced recombinant protein technologies. We will implement modern DNA assembly technologies and incorporate efficient protein production strategies to overcome these technical bottlenecks. The EMB Core will serve as a research, production, and training resource for all four Projects. We will develop robust pipelines for (1) engineering vectors to aid soluble expression and purification for three established expression systems – bacteria, insect cells and human cells, and constructing single-gene and multi-gene expression vectors using advanced DNA assembly techniques, such as BioBrick and Gibson assembly, (2) providing expression systems that facilitate efficient purification of well-behaved, functional DR complexes, e.g. by incorporating cleavable GFP tags to be employed with GFP-binder single-chain nanobody columns, (3) validating protein interactions and partnerships in DR complexes and assemblies, and (4) using streamlined scaled-up expression platforms to support large-scale cell production. We propose three Aims to empower SBDR-5. Aim 1. Meet the molecular biological needs of SBDR. We will generate in-house modified vectors and create expression constructs for expressing single DR proteins and multi-protein complexes for desired host systems. Aim 2. Provide key protein production resources for SBDR. The available resources include large-scale insect cell and bacteria production, selected human cell production for difficult-to-express DR targets, purification of recombinant proteins when needed, and validation of interacting partners in protein complexes. Aim 3. Establish and maintain a Repository/Distribution Center for SBDR. We have amassed a large collection of key reagents and maintained a Repository/Distribution Center. A secure EMB website with a searchable database will be created to streamline access to these resources and allow timely distribution of the requested reagents to the SBDR labs. All four Projects will rely on EMB Core services to support their molecular biological and protein needs. The EMB Core has established close interactions with all Projects during the previous four funding cycles. Ready access to EMB Core’s enabling expertise and resources will synergize all four Projects, avoid duplication of research efforts, and promote active collaborations. The proposed EMB technologies and services will, in concert with the SCB Core, accelerate SBDR-5 research and empower SBDR Projects by facilitating quantitative, high-resolution structural insights for DNA repair machinery functions.