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EMB-ML Expression, Molecular Biology and MacroLab Core

EMB-ML 表达、分子生物学和 MacroLab 核心

基本信息

批准号：
8914503
负责人：
Priscilla K. Cooper
金额：
$ 55.18万
依托单位：
UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至 2016-09-12
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8914503
关键词：
Address Antibodies Archives Back Binding Biochemical Biochemistry Biological Biological Assay Biology Cancer Intervention Cataloging Catalogs Cells Cellular biology Cloning Collaborations Complementary DNA Complex DNA Damage DNA Repair DNA repair protein DNA-Protein Interaction Development Engineering Ensure Escherichia coli Eukaryotic Cell Evaluation Funding Genetic Goals Human Insecta Laboratories Length Link Methodology Molecular Molecular Biology Molecular Conformation Mutagenesis Phenotype Pichia Procedures Production Proteins Protocols documentation Public Health Randomized Reagent Recombinant Proteins Research Research Personnel Research Project Grants Resource Development Resources Saccharomyces cerevisiae Sampling Secure Services Solubility Solutions Structure System Technology Testing Time Validation design domain mapping expression cloning expression vector feeding flexibility genome integrity improved innovative technologies large scale production macromolecular assembly meetings member new technology novel strategies programs protein complex protein expression protein folding protein protein interaction reconstitution repository response scale up vector web site

项目摘要

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.

SBDR3的总体目标是通过对DNA修复机器的结构和功能的了解，实现癌症干预的机械化、预测性生物学。SBDRS面临的主要挑战包括：(1)控制基因组完整性的全长和修饰蛋白质和复合体的有效重组和组装；(2)确定溶液中灵活的多蛋白质复合体的结构；(3)将结构与生化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资助期内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园区内的协作、高通量(HT)生物分子工程设施MacroLab整合在一起，形成新的EMB-ML核心，以满足SBDRS日益增长的需求。合并后的EMB-ML Core将通过提供专职的核心工作人员和集中的资源、方法和试剂来解决SBDRS的挑战，以克服单个研究实验室往往无法克服的不同项目的共同瓶颈。新构建的EMB-ML Core将作为所有六个项目的生产和开发资源，并将为定制和HT构建表达载体、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，在大肠杆菌和昆虫细胞中大规模生产和表达重组蛋白质结构，在特定项目需要时纯化重组蛋白质，以及关键研究试剂的存档。MacroLab将提供快速进行HT克隆、突变和细菌表达测试的补充能力。重要的是，宏观实验室的开发工作将旨在提供新的技术和平台，以进一步提高产量和产量，这也将反馈到教育统筹局的组成部分。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载体构建；3)开发无细胞羟色胺蛋白生产系统。因此，集成的EMB-ML Core提供了现有技术和新技术的强大结合，并有效地提供了试剂，以启动旨在表征瞬时相互作用和动态构象的项目，这些相互作用和动态构象控制对DNA损伤做出反应的多蛋白质复合体的组装和功能。