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

The Problem: Iron Deficiency Over 2 Billion People Live with Iron Deficiency That’s 30% Of the World’s Population

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  Reasonable conditions.  Stabilizes to orig. pH.  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:  Human stomach empties in 4-5 hours  Target: 1.0µm lysis enzyme HOLINS: The Protein Clocks of Bacteriophage Infections Ing-Nang Wang, David L. Smith, and Ry Young Annual Review of Microbiology, Vol. 54: 799 -825

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