creating a red blood cell substitute researchers arthur yu austin day david tulga hannah cole...
TRANSCRIPT
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
Piron
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
Piron
Phenotype of Dead Cells
With ArabinoseWithout Arabinose
Cells Don’t Lyse
Proteins Remain Intact
Empty Sack of Protein
Piron
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