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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

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Presentation Transcript
bactoblood

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

Advisors

John Dueber • Christopher Anderson • Adam Arkin • Jay Keasling

UC BERKELEY

BACTOBLOOD

Creating a Red Blood Cell Substitute

artificial blood substitutes
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 considerations
Human Practice Considerations
  • What makes a system novel and non-obvious?
  • the functional integration of all the devices into a single system (e.g. Bactoblood)

Tension between open source and patentability

Piron

What is patentable: the part or the application of the part?

the combination of parts that provide a function (device)

human practice considerations1
Human Practice Considerations
  • 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 considerations2
Human Practice Considerations
  • What makes a system novel and non-obvious?
  • the functional integration of all the devices into a single system (e.g. Bactoblood)

Conventional Method

Prove novel and non-obvious

Future of Patenting?

Possibilities Unknown

Piron

Piron

vs.

the chassis
The Chassis

Protect Recipient

from E. coli

Protect E.coli from

Immune System

Lipopolysaccharide

(LPS)

K1:O16 capsule

Piron

Pili and Flagella

tonB gene

expression of human hemoglobin
Expression of Human Hemoglobin

DimericHbA

Piron

MonomericHbA

system components
System Components

Heme

Antioxidants

Alpha Hemoglobin Stabilizing Protein

Cytochome b5 / Cytochrome b5 Reductase

Piron

freeze drying
Freeze Drying
  • Bactoblood can be stockpiled and easily transported
  • 2 desiccation devices which prevent cell damage

Trehalose

Hydroxyectoine

Piron

  • 2 genes from e. coli genome
  • Four genes from
  • Streptomyceschrysomallus

Both help cells recover after freeze-drying

freeze drying1
Freeze Drying

Actual Lyophilized Bactoblood

Trehalose

Bactoblood Culture

Piron

the controller
The Controller

Directs copy number and transcription of system devices

Bacterial Artificial Chromosome (single copy)

  • T7 Polymerase
  • pirgenes
  • Iron-inducible promoter

Piron

pSC101 Derived Plasmid

(low copy)

  • Biosynthetic Operons
  • T7 Promoters
  • pir dependent R6K Origin
controller part characterization
Controller Part Characterization

T7 RNA Polymerase

Iron Promoter, yfbE

Piron

  • Only composite part with the weakest rbs and a GTG start
  • codon showed iron-dependent GFP production
copy number device assays
Copy Number Device Assays

GFP Cytometry

As copy number increases, so does the amount of GFP

Low copy number

No pir

Piron

High copy number

Pir genes

Iron-dependent copy number

Pir+Controller

Induced with Iron

No Iron

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

Piron

kill switch growth assays
Kill Switch Growth Assays

# of colonies

Piron

Piron

+ Arabinose

+ Arabinose

- Arabinose

- Arabinose

phenotype of dead cells
Phenotype of Dead Cells

Cells Don’t Lyse

Proteins Remain Intact

Empty Sack of Protein

Without Arabinose

With Arabinose

Piron

making comprehensive systems
MakingComprehensive Systems

Bactoblood Devices

Hemoglobin

Create devices to address specific aspects

Peroxide Damage Control

Heme

Freeze-Drying

Controller

Genetic Kill Switch

acknowledgements
Acknowledgements

The Arkin and Keasling Labs

Kate Spohr, Kevin Costa and Gwyneth Terry

SynBERC

The Camille and Henry Dreyfus Foundation