Creating a Red Blood Cell Substitute 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 John Dueber Christopher Anderson Adam Arkin Jay Keasling

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

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

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

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

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

P iron Expression of Human Hemoglobin Dimeric HbA Monomeric HbA

System Components Alpha Hemoglobin Stabilizing ProteinHemeAntioxidantsCytochome b5 / Cytochrome b5 Reductase P iron

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

TrehaloseBactoblood Culture Actual Lyophilized Bactoblood Freeze Drying P iron

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

The Controller P iron

Controller Part Characterization Iron Promoter, yfbE T 7 RNA Polymerase Only composite part with the weakest rbs and a GTG start codon showed iron-dependent GFP production P iron

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

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

Kill Switch Growth Assays P iron # of colonies - Arabinose + Arabinose - Arabinose + Arabinose P iron

Phenotype of Dead Cells With ArabinoseWithout Arabinose Cells Don’t Lyse Proteins Remain Intact P iron

A Comprehensive System Oxygen Delivery Peroxide Damage Control Survival in Bloodstream Inability to Replicate Universal Compatibility Ability to be Freeze-dried Self-replicating Disease Free y es y es y es y es

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

Patent Timeline 1 yr 3-10 yrs * Avg. 3 yrs 20 yrs from original patent application Provisional Patent Application Filed Provisional app expires and a Utility Patent App. must be filed 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. 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 Team finishes finial touches of Bacto Blood Parts made Public on Registry/ The application of the parts are publicly disclosed. Patent expires. Invention enters public domain. How might synthetic biology be a driver for inventing new modes of industrial practices and partnerships other than the current open source approach?

Swarming Assay P iron Wild Type (with flagella)Chassis (no flagella)

Serum Survival Assay P iron

Oxygen Transport P iron Oxygen Delivery pO2 (Torr) % O2 Saturation P 50 Value 1 AU

Oxygen Transport P iron 25 AU Oxygen Delivery pO2 (Torr) % O2 Saturation P 50 Value 100 AU

3+3+ Problems P iron O2O2 O2O2 O2O2 O2O2 O2O * Doesn’t Work Free Radicals Not Good Superoxide Methemoglobin