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My N.U. P.A.L.

My N.U. P.A.L. N orthwestern U niversity’s P seudomonas a eruginosa L ocator. 1.7 million. Hospital acquired Infections. 170,000. Pseudomonas aeruginosa Infections. Quorum Sensing. R. R. R. Target Gene . Autoinducer gene. Expression. Bacterium.

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My N.U. P.A.L.

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  1. My N.U. P.A.L. Northwestern University’s Pseudomonas aeruginosa Locator

  2. 1.7 million Hospital acquired Infections 170,000 Pseudomonas aeruginosa Infections

  3. Quorum Sensing R R R Target Gene Autoinducer gene Expression Bacterium

  4. Quorum Sensing in Pseudomonas aeruginosa PAI-2 PAI-1 LasR LasR LasR LasR LasR RhlP LasP Las sequence Rhl sequence RhlR RhlR RhlR RhlR RhlR RhlR

  5. Project Goal Biosensor Reliable Fast Simple & Inexpensive

  6. System Overview

  7. Autoinducer Pseudomonas aeruginosa R R R R R Induced Promoter R-protein synthase RBS RBS GFP synthase Constitutive Promoter E. Coli Biosensor

  8. GFP lasR Finding the Parts rhlP lasP RBS rhlR

  9. Parts Submitted 38 parts Designed and Constructed 21 parts Function Validated 19 parts Characterized

  10. Parts Characterized 6 parts Rhl promoter Constructs 5 parts Las Promoter Constructs 4 parts Newly Isolated Promoter Constucts 4 parts Previously Existing Registry Parts

  11. Favorite Parts LasR, PAI-1 BBa_K575024 RhlR, PAI-2 BBa_K575033 RhlR, PAI-2 Newly isolated BBa_K575046

  12. Testing Methods • Overnights • Sample Preparation • Plate Reader

  13. Las Based Detection System L L BBa_K575024 LasR/PAI-1 Induced Promoter RBS GFP Constitutive Promoter RBS LasR

  14. Time Response of LasR/PAI-1 Biosensor No discrimination between different concentrations Autoinducer added No Autoinducer

  15. LasR/PAI-1 Biosensor Exhibits Binary Sensing Flat transfer function Steady State Fluorescence per OD Steady State Fluorescence of the construct does not change in response to varying PAI-1 dosages Autoinducer Concentrations (μM)

  16. Rhl Based Detection System R R BBa_K575033 RhlR/PAI-2 Induced Promoter RBS GFP Constitutive Promoter RBS RhlR

  17. Time Response of RhlR/PAI-2 Biosensor Increasing autoinducer concentration Discriminates between different concentrations Autoinducer added No Autoinducer

  18. RhlR/PAI-2 Biosensor Exhibits Dose-Dependent Response Y=68.234ln(x) + 142.89 R2=0.9322 Steady State Fluorescence per OD Autoinducer Concentrations (μM)

  19. Rhl Based Detection System R R BBa_K575046 (Isolated from Genome) RhlR/PAI-2 Induced Promoter RBS GFP Constitutive Promoter RBS RhlR

  20. Time Response of RhlR/PAI-2 Biosensor Isolated from the Genome Increasing autoinducer concentration Discriminates between different concentrations Autoinducer added No Autoinducer

  21. RhlR/PAI-2 Biosensor Isolated from the Genome Exhibits Dose-Dependent Response Y=33.43ln(x) + 72.293 R2=0.9694 Steady State Fluorescence per OD Exhibited superior discrimination between varying PAI-2 dosages Autoinducer Concentrations (μM)

  22. System Model

  23. Purpose Better understand why the biosensors behave differently in the context of the applications Optimize parameters and achieve the set goal Develop and apply a model to other pathogen sensing constructs or devices

  24. 12 11 10 Ae Ai 3 5 mRNA M CP R D 4 Directly translated each rate to ODEs 2 1 IP 8 13 6 7 System of non-linear equations 9 mRNA GFP

  25. Analysis Sensitivity Identify Critical Parameters Simulation

  26. Qualitative Mathematical Fit to the LasR Biosensor Data 1μM-100μM 0.5μM Arbitrary Concentration of GFP 0.1μM Data No Autoinducer Simulated Time (min)

  27. Qualitative Mathematical Fit to the RhlR Biosensor Data 100μM 50μM 0.5μM 0.1μM 20μM 10μM 5μM Arbitrary Concentration for GFP Data No Autoinducer Simulated Time (min)

  28. 10 Critical Parameters Ai 11 LasR/PAI-1 12 3 Ae 5 ↓RhlR/PAI-2 M R D mRNA 4 CP 2 1 ↑LasR/PAI-1 RhlR/PAI-2 IP 8 RhlR/PAI-2 , IP +ve co-operativity 13 6 7 9 mRNA GFP

  29. Implications Identified system parameters which fluctuated the biosensors’ response Can potentially optimize constructs to “tune” the sensitivity Generalized model which mathematically represents autoinducer detection

  30. Application Proposed Biosensor Schematic Well Photodiode Blue LED Orange filter

  31. Application Swab is inserted into a vial with the biosensor Swab Biosensor fluoresces in the presence of the pathogen

  32. Biowarfare Tear gas Artificial Life Unknown consequences

  33. Dr. Dale Mortensen Nobel laureate in Economics Dr. Keith Tyo Synthetic biologist Human Practices Dr. Laurie Zoloth Bioethicist We promise not only to do the cool research, but to stand by every mistake and every error from now until the future, just like you would stand by profit taking. -Dr. Laurie Zoloth

  34. Results Summary 19 parts characterized The Las based biosensor exhibited a binary response The Rhl based biosensor exhibited a dose dependent response High discrimination between varying autoinducer dosages exhibited by the newly isolated promoter

  35. Initial Goals Fast Simple and Inexpensive Reliable

  36. Future Prospects Future iGEM Projects Clinical Application

  37. Acknowledgements

  38. THANK YOU. Questions?

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