creating a red blood cell substitute researchers arthur yu austin day david tulga hannah cole...
TRANSCRIPT
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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
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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
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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
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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
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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.
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The Chassis
Piron
Protect E.coli from
Immune System
K1:O16 capsule
Pili and Flagella
tonB gene
Protect Recipient
from E. coliLipopolysaccharide
(LPS)
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Piron
Expression of Human Hemoglobin
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System Components
Alpha Hemoglobin Stabilizing ProteinHemeAntioxidantsCytochome b5 / Cytochrome b5 Reductase
Piron
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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
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Trehalose Bactoblood Culture
Actual Lyophilized Bactoblood
Freeze Drying
Piron
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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
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The Controller
Piron
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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
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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
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Piron
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
Piron
0
500000
1000000
1500000
2000000
2500000
# o
f co
lon
ies
- Ara
binose+
Arabinose
- Ara
binose+
Arabinose
Piron
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Phenotype of Dead Cells
With ArabinoseWithout Arabinose
Cells Don’t Lyse
Proteins Remain Intact
Empty Sack of Protein
Piron
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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
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Acknowledgements
The Arkin and Keasling LabsKate Spohr, Kevin Costa and Gwyneth
TerrySynBERCThe Camille and Henry Dreyfus Foundation