THE Phytastic PROBIOTIC

INCREASING THE BIOAVAILABILITY OF IRON IN THE

DIGESTIVE SYSTEM

Northwestern University iGEM 2012

The Northwestern 2012 iGEM Team

THE Phytastic 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 (such

as nuts, grains, and

bran) is chelated by

phytic acid.

Our Mission Statement

To create an inexpensive and

convenient system that increases

the bioavailability of iron in vivo.

THE Phytastic 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 Phytastic 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.

Placed with two strong constitutive promoters from

the Registry.

Producing Phytase: Assay

THE Phytastic 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 Phytastic 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

1000 lysis enzymes/cell

THE Phytastic 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

High School Outreach

Video Collaboration

The rest of the interview can be viewed on our wiki!

THE Phytastic 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 Conclusion

Phytase system produces phytase within the

cell.

Releasing the phytase demonstrates significant

phytase activity.

pH-inducible system demonstrates strong

inducement of both GFP and lysing of cells

when introduced to a low-pH environment.

The Conclusion: Parts

Newly Isolated Obtained from Registry Obtained from 2011

CUHK team

Aspergillus Niger Phytase Constitutive

promoter/RBS/double

terminator

Pgad Promoter

Bacillus Subtilus Phytase Lysis cassette

Citrobakter Braakii Phytase

E. Coli Phytase

Clc-ec1 antiporter

31 parts designed and constructed.

10 parts fully characterized.

The Conclusion: Favorite Parts

BBa_K912025

Promoter/RBS+ C.

Braakii Phytase

BBa_K912034

pH Inducible GFP

System

BBa_K912033

pH Inducible Lysis

System

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 Phytastic!