My N.U. P.A.L. Northwestern University’s Pseudomonas aeruginosa Locator acquired 1.7 million Hospital Infections 170,000 Pseudomonas aeruginosa Infections Quorum Sensing R R R Target Gene Autoinducer gene Expression Bacterium Quorum Sensing in Pseudomonas aeruginosa PAI-1 PAI-2 LasR LasR RhlR LasR LasR LasR RhlR RhlR RhlR RhlP LasP Las sequence Rhl sequence RhlR RhlR Project Goal Biosensor Fast Simple & Reliable Inexpensive System Overview Autoinducer Pseudomonas aeruginosa R R R Induced Promoter R R-protein synthase R RBS Constitutive Promoter RBS GFP synthase E. Coli Biosensor GFP lasR Finding the Parts rhlP RBS lasP rhlR Parts Submitted 38 parts Designed and Constructed 21 parts Function Validated 19 parts Characterized Parts Characterized 6 parts Rhl promoter Constructs 5 parts Las Promoter Constructs Newly Isolated 4 parts Promoter Constucts Existing 4 parts Previously Registry Parts Favorite Parts LasR, PAI-1 BBa_K575024 RhlR, PAI-2 BBa_K575033 RhlR, PAI-2 BBa_K575046 Newly isolated Testing Methods Overnights Sample Preparation Plate Reader Las Based Detection BBa_K575024 System LasR/PAI-1 RBS Induced Promoter GFP Constitutive Promoter RBS LasR L L Time Response of LasR/PAI-1 Biosensor No discrimination between different concentrations Autoinducer added No Autoinducer 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) Rhl Based Detection BBa_K575033 System RhlR/PAI-2 RBS Induced Promoter GFP Constitutive Promoter RBS RhlR R R Time Response of RhlR/PAI-2 Biosensor Discriminates between different concentrations Increasing autoinducer concentration Autoinducer added No Autoinducer 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) Rhl Based Detection BBa_K575046 System (Isolated from Genome) RhlR/PAI-2 RBS Induced Promoter GFP Constitutive Promoter RBS RhlR R R Time Response of RhlR/PAI-2 Biosensor Isolated from the Genome Discriminates between different concentrations Increasing autoinducer concentration Autoinducer added No Autoinducer 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) System Model 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 12 Ae 11 10 5 M Ai Directly translated each rate to ODEs System of non-linear equations mRNA 4 R 2 1 IP D 8 7 GFP 3 13 6 mRNA 9 CP Analysis Sensitivity Identify Critical Parameters Simulation Qualitative Mathematical Fit to the LasR Biosensor Data 0.7 1μM-100μM 0.6 0.5μM Arbitrary Concentration of GFP 0.5 0.1μM 0.4 0.3 0.2 Data 0.1 0 50 100 150 200 Simulated Time (min) 250 300 No Autoinducer Qualitative Mathematical Fit to the RhlR Biosensor Data 100μM 50μM 20μM 4 3.5 3 Arbitrary Concentration 2.5 for GFP 2 10μM 1.5 5μM 1 0.5 0 50 100 150 200 250 Simulated Time (min) 300 0.5μM 0.1μM No Autoinducer Data Critical Parameters LasR/PAI-1 Ae 10 11 12 ↓RhlR/PAI-2 5 M Ai 1 ↑LasR/PAI-1 IP D 8 7 GFP CP 4 R 2 mRNA 3 RhlR/PAI-2 , IP +ve 6 co-operativity mRNA 9 13 RhlR/PAI-2 Implications Identified system parameters which fluctuated the biosensors’ response Can potentially optimize constructs to “tune” the sensitivity Generalized model which mathematically represents autoinducer detection Application Proposed Biosensor Schematic Well Blue LED Photodiode Orange filter Application Swab Swab is inserted into a vial with the biosensor Biosensor fluoresces in the presence of the pathogen Dr. Dale Mortensen Nobel laureate in Economics Dr. Laurie Zoloth Bioethicist Dr. Keith Tyo Synthetic biologist Human Practices 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 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 Initial Goals Fast Simple and Inexpensive Reliable Future Prospects Future iGEM Projects Clinical Application Acknowledgements THANK YOU. Questions?