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THE PROBIOTIC
INCREASING THE BIOAVAILABILITY OF IRON IN THE
DIGESTIVE SYSTEM
Northwestern University iGEM 2012
The Northwestern 2012 iGEM Team
THE PROBIOTIC
1. The Problem: Iron Deficiency
2. The Solution: Phytase
3. The Implementation
1. Producing Phytase
2. Delivering Phytase
4. The Model
5. The Ethics
6. The Conclusion
Over 2 Billion People Live with Iron Deficiency
That’s 30% Of the World’s Population
The Problem: Iron Deficiency
The Problem: Iron Deficiency
Iron deficiency
has worldwide
prevalence
among all
populations
The Symptoms of Iron Deficiency
Iron-Deficiency Anemia
Extreme Fatigue
Shortness of Breath
Impaired Immune
System
Impaired Mental
Function
Eventually Death
A Cause: Phytic Acid
Iron is usually present
in diet, but it is not
readily available for
absorption.
The iron that is found
in certain plants is
chelated by phytic
acid.
Our Mission Statement
To create an inexpensive and
convenient system that increases
the bioavailability of iron in vivo.
THE PROBIOTIC
1. The Problem: Iron Deficiency
2. The Solution: Phytase
3. The Implementation
1. Producing Phytase
2. Delivering Phytase
4. The Model
5. The Ethics
6. The Conclusion
The Solution: Phytase
Phytase: an enzyme that breaks down phytic acid.
Cleaves phosphate groups from phytic acid,
unbinding iron (and other nutrients)!
Delivering Phytase
Develop a probiotic that
will release phytase.
Fermented milk products
native to African and South
Asian diets.
These areas have a high
concentration of people
with iron deficiency.
THE PROBIOTIC
1. The Problem: Iron Deficiency
2. The Solution: Phytase
3. The Implementation
1. Producing Phytase
2. Delivering Phytase
4. The Model
5. The Ethics
6. The Conclusion
Producing Phytase: Design
Successfully isolated phytase from Aspergillus niger,
Bacillus subtilis, Citrobacter braakii, and Escherichia
coli.
Tested with two strong constitutive promoters from the
Registry.
Producing Phytase: Assay
THE PROBIOTIC
1. The Problem: Iron Deficiency
2. The Solution: Phytase
3. The Implementation
1. Producing Phytase
2. Delivering Phytase
4. The Model
5. The Ethics
6. The Conclusion
Delivering Phytase: Design
In order to release phytase into the stomach, the
probiotic must lyse after ingestion.
Delivering Phytase: Design
Stomach has a high HCl content (low pH).
ClC antiporter exchanges external chloride ions for
internal protons, increasing concentration of
intracellular chloride ions.
Pgad promoter detects increased concentration of
intracellular chloride ions, activating lysis cassette.
Pgad/Lysis at pH7
Pgad/Lysis at pH2
Delivering Phytase: Alternate Part
Cloned and created a part that would produce GFP
instead of lysing.
Originally used for testing purposes.
Can also be used simply as a low-pH detection
system.
Limitations: requires extracellular chloride.
Referred to simply as “Pgad/GFP.”
Pgad/GFP at pH 7
Pgad/GFP at pH 2
THE PROBIOTIC
1. The Problem: Iron Deficiency
2. The Solution: Phytase
3. The Implementation
1. Producing Phytase
2. Delivering Phytase
4. The Model
5. The Ethics
6. The Conclusion
Phytastic System Model
Goal: Address the issue of
Is the Phytastic system
plausible?
Model Design:
Simulate Phytastic cells
entering the stomach as a
system of ODEs.
ODE Model
Antiporter Component Pgad/GFP component
Model Results
Recovers quickly
Stabilizes to orig. pH.
Reasonable conditions.
Comparable to literature.
Slonczewksi, J. L., R. M. Macnab, J. R. Alger, and A. M. Castle. 1982. Effects of pH and repellent tactic stimuli on protein methylation levels in
Escherichia coli. J. Bacteriol. 152:384-399.
Model Results
Assess goal of plausibility:
Constraints:
HOLINS: The Protein Clocks of Bacteriophage Infections Ing-Nang Wang, David L. Smith, and Ry Young
Annual Review of Microbiology, Vol. 54: 799 -825
Human stomach empties in 4-5 hours
Target: 1.0µm lysis enzyme
THE PROBIOTIC
1. The Problem: Iron Deficiency
2. The Solution: Phytase
3. The Implementation
1. Producing Phytase
2. Delivering Phytase
4. The Model
5. The Ethics
6. The Conclusion
The Ethics: High School Outreach
The Ethics: Video Collaboration
The rest of the interview can be viewed on our wiki!
The Ethics: Questions
Do the cultures we target accept Genetically
Modified Organisms (GMOs) in their diet?
Why do these cultures not accept GMOs?
How do we resolve this issue and make GMOs more
acceptable to the general public?
THE PROBIOTIC
1. The Problem: Iron Deficiency
2. The Solution: Phytase
3. The Implementation
1. Producing Phytase
2. Delivering Phytase
4. The Model
5. The Ethics
6. The Conclusion
Our Mission Statement
To create an inexpensive and
convenient system that increases
the bioavailability of iron in vivo.
The Conclusion
Probiotic that does not
disrupt habits or the
native culture and
easily integrates into
present diets.
The Conclusion
Phytase system
produces phytase
within the cell and
cleaves phosphorus
groups from phytic
acid.
The Conclusion
Modular pH-inducible
system demonstrates
strong inducement of
both GFP and lysing
of cells when
introduced to a low-
pH environment.
The Conclusion: Future Work
We plan on:
Changing the chassis from E. coli to a lactic
acid bacteria
Produce a fermented milk product with the
bacteria
Acknowledgements
Special thanks to:
Department of Biological Sciences
Jewett Lab graduate students
2011 Hong Kong-CUHK team for taking time to send us
a part they characterized that was not in the Parts
Registry!
Thank You
Be