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BACTOBLOOD

Researchers Arthur Yu • Austin Day • David Tulga • Hannah Cole • Kristin Doan • Kristin Fuller • Nhu Nguyen • Samantha Liang • Vaibhavi Umesh • Vincent Parker Teaching Assistants Amin Hajimorad • Farnaz Nowroozi • Rickey Bonds Advisors

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BACTOBLOOD

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  1. ResearchersArthur Yu • Austin Day • David Tulga • Hannah Cole • Kristin Doan • Kristin Fuller • Nhu Nguyen • Samantha Liang • Vaibhavi Umesh • Vincent Parker Teaching AssistantsAmin Hajimorad • Farnaz Nowroozi • Rickey Bonds Advisors John Dueber • Christopher Anderson • Adam Arkin • Jay Keasling UC BERKELEY BACTOBLOOD Creating a Red Blood Cell Substitute

  2. Artificial Blood Substitutes The Need • Supply shortage, especially in • developing countries • PFC limitations • HBOC limitations Benefits of Bactoblood • Universally compatible • Disease-free • Inexpensive • Ability to be stored for a prolonged period • Rapid production in emergency situations

  3. Human Practice IP Considerations What makes Bactoblood novel and non-obvious? the functional integration of all the devices into a single system What is patentable: the part or the application of the part? the combination of parts that provide a function (device) Piron

  4. Human Practices IP Considerations What makes Bactoblood novel and non-obvious? the functional integration of all the devices into a single system Piron

  5. Human Practices IP Considerations What makes Bactoblood novel and non-obvious? the functional integration of all the devices into a single system Patentability of Bactoblood may depend on what aspects of the invention are claimed in a patent application & how it is worded. Piron Piron

  6. The Chassis Protect Recipient from E. coli Protect E.coli from Immune System Lipopolysaccharide (LPS) K1:O16 capsule Piron Pili and Flagella tonB gene

  7. Expression of Human Hemoglobin Dimeric HbA Piron Monomeric HbA

  8. System Components Heme Antioxidants Alpha Hemoglobin Stabilizing Protein Cytochome b5 / Cytochrome b5 Reductase Piron

  9. Freeze Drying • Bactoblood can be stockpiled and easily transported • 2 desiccation devices which prevent cell damage Trehalose Hydroxyectoine Piron • 2 genes from e. coli genome • Four genes from • Streptomyces chrysomallus Both help cells recover after freeze-drying

  10. Freeze Drying Actual Lyophilized Bactoblood Trehalose Bactoblood Culture Piron

  11. The Controller Directs copy number and transcription of system devices Bacterial Artificial Chromosome (single copy) • T7 Polymerase • pir genes • Iron-inducible promoter Piron pSC101 Derived Plasmid (low copy) • Biosynthetic Operons • T7 Promoters • pir dependent R6K Origin

  12. The Controller Piron

  13. Controller Part Characterization T7 RNA Polymerase Iron Promoter, yfbE Piron • Only composite part with the weakest rbs and a GTG start • codon showed iron-dependent GFP production

  14. Copy Number Device Assays GFP Cytometry As copy number increases, so does the amount of GFP Low copy number No pir Piron High copy number Pir genes Iron-dependent copy number Pir+Controller Induced with Iron No Iron

  15. Genetic Kill Switch • Prevents chance of infection or unwanted proliferation • When induced, cells degrade their own DNA Piron

  16. Kill Switch Growth Assays # of colonies Piron Piron + Arabinose + Arabinose - Arabinose - Arabinose

  17. Phenotype of Dead Cells Cells Don’t Lyse Proteins Remain Intact Empty Sack of Protein Without Arabinose With Arabinose Piron

  18. A Comprehensive System Bactoblood Oxygen Delivery Peroxide Damage Control Survival in Bloodstream Inability to Replicate • yes • yes • yes • yes • Universal Compatibility • Ability to be Freeze-dried • Self-replicating • Disease Free Red Blood Cells www.cleoconference.org

  19. Acknowledgements The Arkin and Keasling Labs Kate Spohr, Kevin Costa and Gwyneth Terry SynBERC The Camille and Henry Dreyfus Foundation

  20. Patent Timeline When Bacto Blood’s patent issues, the patent holders may exclude others from use of the invention and may license Bacto Blood to others for use. Team finishes finial touches of Bacto Blood During this time patent app may be allowed or rejected. If rejected team re-writes the claim in patent app and sends it to patent examiner for further examination 1 yr 1 yr 3-10 yrs *Avg. 3 yrs 20 yrs from original patent application Provisional app expires and a Utility Patent App. must be filed Patent expires. Invention enters public domain. Provisional Patent Application Filed Parts made Public on Registry/ The application of the parts are publicly disclosed. How might synthetic biology be a driver for inventing new modes of industrial practices and partnerships other than the current open source approach?

  21. Swarming Assay Wild Type (with flagella) Chassis (no flagella) Piron

  22. Serum Survival Assay Piron

  23. Oxygen Transport Oxygen Delivery 1 AU P50 Value Piron % O2 Saturation pO2 (Torr)

  24. Oxygen Transport Oxygen Delivery 100 AU 25 AU P50 Value Piron % O2 Saturation pO2 (Torr)

  25. O2 Problems Superoxide Methemoglobin Doesn’t Work * Piron Free Radicals O2 2+ 3+ Not Good O2 O2 O2

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