EMB-ML Expression, Molecular Biology and MacroLab Core

SBDR3的总体目标是通过对DNA修复机器的结构和功能的理解，实现癌症干预的机制和预测生物学。SBDRS面临的主要挑战包括：(1)有效地重组和组装全长和修饰的蛋白质和复合物，以控制基因组的完整性；(2)确定溶液中柔性多蛋白复合物的结构；(3)将结构与生物化学和细胞表型联系起来。我们建议将劳伦斯伯克利国家实验室在前两个SBDR资助期间开发的非常成功的表达和分子生物学核心与加州大学伯克利分校校园内的高通量（HT）生物分子工程设施MacroLab结合起来，形成一个新的EMB-ML核心，以满足SBDRS不断增长的需求。合并后的EMB-ML核心将通过提供专门的核心人员和集中的资源、方法和试剂来解决SBDRS的挑战，以克服不同项目常见的瓶颈，这些瓶颈通常是单个研究实验室无法克服的。新构建的EMB-ML核心将作为所有六个项目的生产和开发资源，并将为表达载体的定制和高温构建、大规模细胞生产和蛋白质相互作用验证提供管道。EMB组件将提供合理的设计和定制的表达载体构建，蛋白质-蛋白质和蛋白质- dna相互作用的验证，重组蛋白构建物在大肠杆菌和昆虫细胞中的大规模生产和表达，特定项目需要时的重组蛋白纯化，以及关键研究试剂的存档。MacroLab将提供进行快速HT克隆、诱变和细菌表达检测的补充能力。重要的是，MacroLab的发展努力将旨在提供新的技术和平台，以进一步提高产量和产量，这也将反馈到EMB组件中。创新技术包括1)在真核细胞中创建自动化（96孔）克隆和表达平台；2)实现半自动多顺反子和多启动子向量构建；3)开发无HT细胞蛋白生产系统。因此，集成的EMB-ML Core提供了现有技术和新技术的强大混合，并有效地提供试剂，以启动旨在表征瞬态相互作用和动态构象的项目工作，这些构象控制多蛋白复合物对DNA损伤的组装和功能。

The overall goal of SBDR3 is to achieve mechanistic, predictive biology for cancer interventions through structural and functional understanding of DNA repair machines. The major challenges faced by SBDRS include: (1) efficient reconstitution and assembly of full-length and modified proteins and complexes that control integrity of the genome, (2) determining structures of flexible multi-protein complexes in solution, and (3) linking structures to biochemistry and cellular phenotypes. We propose to integrate the highly successful Expression and Molecular Biology Core that was developed at Lawrence Berkeley National Lab during the two previous SBDR funding periods with the MacroLab, a collaborative, high throughput (HT) biomolecular engineering facility on the UC Berkeley campus, to form a new EMB-ML Core for meeting the increased needs of SBDRS. The combined EMB-ML Core will address SBDRS challenges by providing dedicated Core staff and centralized resources, approaches, and reagents to overcome the bottlenecks common to different projects that are often insurmountable for a single research lab. The newly structured EMB-ML Core will serve as both a production and development resource for all six Projects, and will provide a pipeline for customized and HT construction of expression vectors, large-scale cell production, and protein interaction validation. The EMB component will provide rational design and customized expression vector construction, validation of protein-protein and protein-DNA interactions, large-scale production and expression of recombinant protein constructs in E. coli and insect cells, purification of recombinant proteins when needed for particular projects, and archiving of critical research reagents. The complementary ability to carry out rapid HT cloning, mutagenesis, and bacterial expression testing will be provided by the MacroLab. Importantly, developmental efforts in the MacroLab will be aimed at providing new technologies and platforms for further increases in throughput and yields, which will also be fed back into the EMB component. Innovative technologies include 1) creating automated (96-well) cloning and expression platforms in eukaryotic cells; 2) implementing semi-automatic polycistronic and polypromoter vector construction; and 3) developing a HT cell-free protein production system. The integrated EMB-ML Core thus offers a robust blend of established and new technologies, and efficiently provides reagents to jump-start Project efforts aimed at characterizing transient interactions and dynamic conformations that control the assembly and function of multi-protein complexes responding to DNA damage.