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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 of large number of biological

  1. Automated retrieval of viable microorganism samples: the IcePick Jef Boeke Funding: NIH Roadmap Technology Center for Networks and Pathways

  2. Learning objectives • 1. Challenges associated with storage/retrieval of 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 optimal storage conditions for long term viability, and reduces the tedium associated with mundane activities associated with biobanking

  3. Themes • Technology, technology, technology • Histones and chromatin • Ubiquitin etc. • Most heavily modified • Unique proteinaceous proteins PTM family QuickTime™ and a • Profound biological TIFF (Uncompressed) decompressor • Profound biological are needed to see this picture. effects of Lysine PTMs effects on many • Wide variety of Lysine pathways PTMs • Pickart, Cohen, Berger, QuickTime™ and a • Berger, Biggins, TIFF (LZW) decompressor Matunis, Meluh, are needed to see this picture. Onyango, Wolberger, Shilatifard, Varmus, Shilatifard, Krogan, Wolberger Verreault, Zhang

  4. Technology cores Function chips 1 Function chips 2 Zhu Boeke Computation/ Modeling Bader Mass Spec 2 Cotter QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Chemistry Cole Mass Spec 1 Pandey

  5. Heng Zhu lab - “Function chips” Protein microarrays to probe PTM networks and pathways through binding and enzyme activity studies GST:P1 GST:P2 GST:P3 Proteins in 35% Glycerol Protein-Protein Protein-modification Other

  6. Boeke lab Function chips Technology 2 •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

  7. New unique resources External resources (Boeke lab) Histone mutant collection v. 1.0 (approx 250 mutants) E. coli ORFs; H. Mori; via Burnham’s TCNP Approx 4,000 clones Histone H3/H4 mutant collection v. 2.0 ~1000 mutants Human ORFs (and planned subclones); Invitrogen Approx 80,000 clones Ts mutants in essential Lysine modification genes Human and Mouse shRNAs; TRC Approx 150,000 clones Yeast deletion collections, NHGRI funded Approx 33,000 clones Ts mutants in other essential genes; Phil Hieter, UBC; NCI funded resource Total clones to manage: Approx 6,000 clones >300,000 clones This ‘n that The IcePick TM Frozen Resource Approx 30,000 clones Distribution System

  8. 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 Quic IFF (Uncomp are needed v. 1.0 - Sharon

  9. Starting point: the Biophile Key Features: Commercially available •-80˚C freezer / dry N 2 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

  11. 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 only part of the problem

  14. (Show movie)

  15. Input/Output • An online order is placed on • Requested samples are a secure web server picked one at a time into a destination plate as specified • Requested items are found in a database of locations in • Requested materials are bar-coded source plates distributed to requestor • A spreadsheet is generated • Number of times a source specifying which bacteria or well is tapped is recorded; samples are required and “best-sellers” are identified where those samples will be and replicated deposited in a destination • Full audit trail of who plate accessed what, when

  16. Microbiological Performance A B A Yeast in YPD medium B Bacteria in LB medium C 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

  17. 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…

  18. Drivers for Automated Biobanking QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. From J. Comley, Drug Discovery World, Summer 2007

  19. Some biological sample types required to be stored in a biobank; preferred storage temperatures QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. From J. Comley, Drug Discovery World, Summer 2007

  20. Mean storage time in automated biobanks at various temperatures Mean storage time (years) QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. -150˚C -80˚C -20˚C From J. Comley, Drug Discovery World, Summer 2007

  21. 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 QuickTime™ and a QuickTime™ and a QuickTime™ and a TIFF (LZW) decompressor TIFF (LZW) decompressor TIFF (LZW) decompressor are needed to see this picture. are needed to see this picture. are needed to see this picture.

  22. 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

  23. 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.

  24. Acknowledgements • Alan Shunliu Long • Heng Zhu • Min Li

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