Download Jam07_UCBerkeley

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
Creating a Red Blood Cell Substitute
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
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
Human Practices IP Considerations
What makes Bactoblood novel and non-obvious?
the functional integration of all the devices into a single system
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Human Practices IP Considerations
What makes Bactoblood novel and non-obvious?
the functional integration of all the devices into a single system
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Patentability of Bactoblood
may depend on
what aspects of the invention are claimed in a
patent application & how it is worded.
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The Chassis
Protect Recipient
from E. coli
Protect E.coli from
Immune System
Lipopolysaccharide
(LPS)
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Pili and Flagella
tonB gene
K1:O16 capsule
Expression of Human Hemoglobin
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System Components
Cytochome
Alpha Hemoglobin
b5Antioxidants
/ Cytochrome
Heme
Stabilizing
b5 Reductase
Protein
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Freeze Drying
• Bactoblood can be stockpiled and easily transported
• 2 desiccation devices which prevent cell damage
Trehalose
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• 2 genes from e. coli genome
Hydroxyectoine
• Four genes from
Streptomyces chrysomallus
Both help cells recover after freeze-drying
Freeze Drying
Trehalose
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Bactoblood Culture
Actual Lyophilized
Bactoblood
The Controller
Directs copy number and transcription of system devices
pSC101
Derived
Plasmid
(low copy)
• T7 Polymerase
• pir genes
• Iron-inducible promoter
Bacterial
Artificial
Chromosome
(single copy)
• Biosynthetic Operons
• T7 Promoters
• pir dependent R6K Origin
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The Controller
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Controller Part Characterization
T7 RNA Polymerase
Iron Promoter, yfbE
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• Only composite part with the weakest rbs and a GTG start
codon showed iron-dependent GFP production
Copy Number Device Assays
GFP Cytometry
As copy number increases, so does the amount of GFP
Low copy number
No pir
High copy number
Pir genes
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Iron-dependent
copy number
Pir+Controller
Induced with Iron
No Iron
Genetic Kill Switch
• Prevents chance of infection or unwanted proliferation
• When induced, cells degrade their own DNA
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Kill Switch Growth Assays
2500000
# of colonies
2000000
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1500000
1000000
500000
0
Phenotype of Dead Cells
Proteins
Cells Don’t
Remain
Lyse Without Arabinose
Intact
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With Arabinose
A Comprehensive System
www.cleoconference.org
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
Acknowledgements
The Arkin and Keasling Labs
Kate Spohr, Kevin Costa and Gwyneth Terry
SynBERC
The Camille and Henry Dreyfus Foundation
Patent Timeline
Team finishes
finial touches of
Bacto Blood
1 yr
1 yr
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
3-10 yrs *Avg. 3 yrs
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.
20 yrs from original patent application
Provisional app expires and a Utility Patent
App. must be filed
Provisional Patent Application Filed
Patent expires. Invention
enters public domain.
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?
Swarming Assay
Wild Type (with flagella)
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Chassis (no flagella)
Serum Survival Assay
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Oxygen Transport
Oxygen Delivery
1 AU
P50 Value
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99
% O2 Saturation
Cellular Hemoglobin
Concentration
1
Functional
Non-Functional
pO2 (Torr)
Oxygen Transport
Oxygen Delivery
100
25 AUAU
Cellular Hemoglobin
Concentration
% O2 Saturation
P50 Value
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99
1
Functional
Non-Functional
pO2 (Torr)
Problems
Superoxide
Methemoglobin
Doesn’t Work
*
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Free Radicals
Not Good
2
3+