THE PROBIOTIC INCREASING THE BIOAVAILABILITY OF IRON IN THE - - PowerPoint PPT Presentation

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

• f 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