FLEXGene Consortium

Tools for Manipulating the Proteome

FLEXGene Repository

Introduction

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.

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

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

To accelerate the study of protein function, we need a gold-standard

cDNA repository

Proteomics

Abundance-based

Identify & Quantify

Function-based

Express & Examine

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

Enabling Technologies

Informatics

–Genome sequencing projects

High-throughput technologies

–DNA sequencing

–Liquid handling

–Storage/retrieval

Recombinational Cloning

Traditional DNA Subcloning

Traditional DNA Subcloning

• Individualized strategy required for every gene

• Days to weeks to obtain subclones

• Too inefficient for automation

Moving Genes by Recombination

Mix two plasmids and enzyme

Mix two plasmids and enzymeOnly the Desired Product Survives

Moving Genes by Recombination

• One universal strategy to move cDNAs to any vector

• Single step, 1 hour reaction• High-throughput• Near 100% efficient, no

mutations

Traditional

Recombinational

Shortens Time from Idea to Experiments

Enables High-Throughput Functional Assays

Building layers of information about proteins

n c n c p c c n

A B A X Y Z C L

– – – + – – – –

– – – – – – – –

+ – + – – – – –

1 2 1 2 7 6 1 1

Localization

Interactors

Assay #1

Assay #2

Assay #3

Purification method