1. FLEXGene Consortium Tools for Manipulating the Proteome

2. FLEXGene Repository Introduction

3. The human genome project will continue to be a fundamental resource for all biological and biomedical research. We believe that access to a repository representing the entire proteome is the next example of a needed common resource.

4. Consortium Mission To catalyze a new era of biological and biomedical research by creating a comprehensive gold-standard cDNA repository that enables protein expression in all experimental formats and at any chosen scale

5. Executive Project Overview Organization Public/private partnership Seeking 8-10 industrial members Managed under NIH umbrella Organization Public/private partnership Seeking 8-10 industrial members Managed under NIH umbrella Deliverables cDNA clones representing 20,000 human genes Fully sequence-verified Gold standard Protein expression-ready in any context Multiple reputable distributors Long term quality control process Freedom to operate Deliverables cDNA clones representing 20,000 human genes Fully sequence-verified Gold standard Protein expression-ready in any context Multiple reputable distributors Long term quality control process Freedom to operate

6. To accelerate the study of protein function, we need a gold-standard cDNA repository

7. Proteomics Abundance-based Identify & Quantify Function-based Express & Examine

8. A perfect repository would be… Comprehensive – Ultimate goal should be at least one clone per mRNA Addressable – Clones should be indexed to enable the rapid assembly of user-defined subsets Flexible – Cloning technology should enable easy transfer into any vector Expression-ready – Clones should capture exact coding regions and be amenable to addition of fusion tags Highest standard – All clones should be sequence-verified Affordable – Clones should be available at an acceptable cost

9. Enabling Technologies Informatics –Genome sequencing projects High-throughput technologies –DNA sequencing –Liquid handling –Storage/retrieval Recombinational Cloning

10. Traditional DNA Subcloning

11. Individualized strategy required for every gene Days to weeks to obtain subclones Too inefficient for automation

12. Moving Genes by Recombination Mix two plasmids and enzyme

13. Only the Desired Product Survives Moving Genes by Recombination

14. One universal strategy to move cDNAs to any vector Single step, 1 hour reaction High-throughput Near 100% efficient, no mutations Traditional Recombinational

15. Shortens Time from Idea to Experiments

16. Enables High-Throughput Functional Assays

17. Building layers of information about proteins ncncpccn ABAXYZCL –––+–––– – ––––––– +–+––––– 12127611 Localization Interactors Assay #1 Assay #2 Assay #3 Purification method