EMB-ML Expression, Molecular Biology and MacroLab Core

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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.