Jef Boeke Funding: NIH Roadmap Technology Center for Networks and - - PowerPoint PPT Presentation

Automated retrieval of viable microorganism samples: the IcePick

Jef Boeke

Funding: NIH Roadmap Technology Center for Networks and Pathways

Learning objectives

• 1. Challenges associated with storage/retrieval
• f large number of biological specimens
• 2. Current commercially available storage and

retrieval systems will be described

• 3. Creating optimal storage conditions for rapid

retrieval, minimizing expensive storage space, and how to "cherry pick" selected specimens from a biorepository

• 4. Automation can assure that specimens are

documented and databased, maintained under

• ptimal storage conditions for long term

viability, and reduces the tedium associated with mundane activities associated with biobanking

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Themes

• Histones and chromatin
• Most heavily modified

proteins

• Profound biological

effects of Lysine PTMs

• Wide variety of Lysine

PTMs

• Berger, Biggins,

Onyango, Wolberger, Shilatifard, Krogan, Verreault, Zhang

• Ubiquitin etc.
• Unique proteinaceous

PTM family

• Profound biological

effects on many pathways

• Pickart, Cohen, Berger,

Matunis, Meluh, Shilatifard, Varmus, Wolberger

• Technology, technology, technology

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Technology cores

Mass Spec 1 Pandey Function chips 2 Boeke Chemistry Cole Computation/ Modeling Bader

Function chips 1 Zhu

Mass Spec 2 Cotter

Heng Zhu lab - “Function chips”

Protein microarrays to probe PTM networks and pathways through binding and enzyme activity studies

Protein-Protein Protein-modification Other

Proteins in 35% Glycerol

GST:P1 GST:P2 GST:P3

• Technology: “SLAM” synthetic lethality analyzed by microarray
• Highly parallel DNA chip/molecular barcode method for

identifying genetic interactions with “query gene” or gene of interest

• Technology adapted to studies of essential genes via Ts

mutants (like many genes involved in ubiquitylation) or for special alleles such as non-modifiable alleles in which lysine is substituted by another sidechain

• Adapting technology to global analyses of histone mutants

Boeke lab Function chips Technology 2

New unique resources (Boeke lab)

Histone mutant collection v. 1.0 (approx 250 mutants) Histone H3/H4 mutant collection v. 2.0 ~1000 mutants Ts mutants in essential Lysine modification genes

The IcePickTM Frozen Resource Distribution System Total clones to manage: >300,000 clones

Human ORFs (and planned subclones); Invitrogen Approx 80,000 clones

External resources

• E. coli ORFs; H. Mori; via Burnham’s TCNP

Approx 4,000 clones Human and Mouse shRNAs; TRC Approx 150,000 clones Ts mutants in other essential genes; Phil Hieter, UBC; NCI funded resource Approx 6,000 clones Yeast deletion collections, NHGRI funded Approx 33,000 clones This ‘n that Approx 30,000 clones

Quic IFF (Uncomp are needed

• v. 1.0 - Sharon

The problem: suppose you want 55 specific ORF clones, 226 specific shRNAs, or 192 yeast knockout strains (tomorrow…)? Note: they are all frozen away in 96- or 384-well plates, somewhere or other The solution: you need a microbiologically sound, efficient and error-free retrieval system

Starting point: the Biophile

Key Features: Commercially available

• -80˚C freezer / dry N2 frost-free environment
• Complete sample tracking with bar-coding system
• Multiwell plates - v. economical, no decapping issues
• Holds >900 96- or 384-well plates
• >384,000 total samples per unit
• Automatically delivers any plate in storage via a touch

screen interface on the front door; or via computer

• Delivers any individual plate in the unit within 1 minute
• Complete hands free operation of plates
• Upright design saves lab space compared to

competing units

• Capital cost about 10-15X the cost of a similarly sized

conventional -80X freezer but running expenses similar

What the Biophile can’t do

• Deliver more than one plate at a time
• Delid plates
• It cannot identify and sample the wells of

interest (eg find well H 17)

• This is the most painful and error-prone

aspect of the sample retrieval process; hence, the existing automation solves

• nly part of the problem

(Show movie)

Input/Output

• An online order is placed on

a secure web server

• Requested items are found

in a database of locations in bar-coded source plates

• A spreadsheet is generated

specifying which bacteria or samples are required and where those samples will be deposited in a destination plate

• Requested samples are

picked one at a time into a destination plate as specified

• Requested materials are

distributed to requestor

• Number of times a source

well is tapped is recorded; “best-sellers” are identified and replicated

• Full audit trail of who

accessed what, when

Microbiological Performance

A B C

A Yeast in YPD medium B Bacteria in LB medium C Heat-map, bacterial growth

• No cross contamination
• Transfers 1 to 5 µL
• Transient thawing with heated pin preferable to thawing entire plate
• 100% of wells sampled produce living cells in destination plates
• So far, no need to “reload” new source plates; viability preserved

What does the medical community need?

• Blood samples
• Urine and other liquid samples
• gDNA samples
• Tissue samples
• All of these samples are of medical

interest

• With minimal retooling, an IcePick can

help…

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From J. Comley, Drug Discovery World, Summer 2007

Drivers for Automated Biobanking

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From J. Comley, Drug Discovery World, Summer 2007

Some biological sample types required to be stored in a biobank; preferred storage temperatures

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From J. Comley, Drug Discovery World, Summer 2007

Mean storage time in automated biobanks at various temperatures

• 150˚C
• 80˚C
• 20˚C

Mean storage time (years)

What else is out there?

• Tube-based systems - you still have to

unscrew the caps!

• Room-sized systems - pricy and bulky!
• Nothing else offers automated picking

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What is next?

• Genomic/plasmid/PCR product DNA

sampling and distribution

• Blood/urine samples for proteomic

genomic and other biomarker studies

• Tissue samples
• Downsizing the unit
• One unit serving multiple Biophiles

Take home messages

• 1. Maintaining specimens at -80˚C can

assure long term storage with minimal degradation in quality

• 2. Automation can be useful to store,

retrieve, aliquot, and document biobanking activities

• 3. Specimen retrieval is enhanced by pulling

specimens directly from a frozen microplate

• 4. There is an optimal mix of parent,

daughter, and aliquot specimens that maximizes research throughput and return on investment.

Acknowledgements

• Alan Shunliu Long
• Heng Zhu
• Min Li