UC BERKELEY

BACTOBLOOD

Creating a Red Blood Cell Substitute

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

AdvisorsJohn 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 Considerations

What is patentable: the part or the application of the part?the combination of parts that provide a function (device)

What makes a system novel and non-obvious?the functional integration of all the devices into a single system

(e.g. Bactoblood)

Piron

Tension between open source and patentability

Piron

What makes a system novel and non-obvious?the functional integration of all the devices into a single

system (e.g. Bactoblood)

Human Practice Considerations

Piron

What makes a system novel and non-obvious?the functional integration of all the devices into a single system

(e.g. Bactoblood)

Piron

Human Practice Considerations

Conventional MethodProve novel and

non-obvious

Future of Patenting?

Possibilities Unknown

vs.

The Chassis

Piron

Protect E.coli from

Immune System

K1:O16 capsule

Pili and Flagella

tonB gene

Protect Recipient

from E. coliLipopolysaccharide

(LPS)

Piron

Expression of Human Hemoglobin

System Components

Alpha Hemoglobin Stabilizing ProteinHemeAntioxidantsCytochome b5 / Cytochrome b5 Reductase

Piron

Freeze Drying

• Bactoblood can be stockpiled and easily transported• 2 desiccation devices which prevent cell damage

HydroxyectoineTrehalose

• Four genes from Streptomyces chrysomallus

• 2 genes from e. coli genome

Both help cells recover after freeze-drying

Piron

Trehalose Bactoblood Culture

Actual Lyophilized Bactoblood

Freeze Drying

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The Controller

Piron

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

Piron

Controller Part Characterization

Iron Promoter, yfbE

T7 RNA Polymerase

• Only composite part with the weakest rbs and a GTG start codon showed iron-dependent GFP production

Piron

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

Piron

Piron

Genetic Kill Switch

• Prevents chance of infection or unwanted proliferation• When induced, cells degrade their own DNA

Kill Switch Growth Assays

Piron

0

500000

1000000

1500000

2000000

2500000

# o

f co

lon

ies

- Ara

binose+

Arabinose

- Ara

binose+

Arabinose

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Phenotype of Dead Cells

With ArabinoseWithout Arabinose

Cells Don’t Lyse

Proteins Remain Intact

Empty Sack of Protein

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Making Comprehensive Systems

Bactoblood DevicesHemoglobin

Peroxide Damage Control

Heme

Freeze-Drying

Controller

Genetic Kill Switch

Create devices to address specific aspects

Acknowledgements

The Arkin and Keasling LabsKate Spohr, Kevin Costa and Gwyneth

TerrySynBERCThe Camille and Henry Dreyfus Foundation