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A Synchronized Oscillator with Quorum Sensing

A Synchronized Oscillator with Quorum Sensing. Presented by Susan Westfall, Tim Johnstone, James Gabriels and Avi Biswas. The Quorum Sensing System. Quorum Sensing Small Diffusible Molecules Regulate Gene Expression Cell-to-Cell Communication Coordination of Cellular Networks.

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A Synchronized Oscillator with Quorum Sensing

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  1. A Synchronized Oscillator with Quorum Sensing Presented by Susan Westfall, Tim Johnstone, James Gabriels and Avi Biswas

  2. The Quorum Sensing System • Quorum Sensing • Small Diffusible Molecules • Regulate Gene Expression • Cell-to-Cell Communication • Coordination of Cellular Networks

  3. Applications Simulating the Oscillator with the Repressilator in E.Coli

  4. Applications Understanding Circadian Rhythms Simulating the Oscillator with the Repressilator in E.Coli

  5. Applications Understanding Circadian Rhythms Simulating the Oscillator with the Repressilator in E.Coli

  6. Applications Understanding Circadian Rhythms Simulating the Oscillator with the Repressilator in E.Coli

  7. Applications Understanding Circadian Rhythms Simulating the Oscillator with the Repressilator in E.Coli

  8. Applications Understanding Circadian Rhythms Simulating the Oscillator with the Repressilator in E.Coli

  9. Our Inspiration • McMillan et al. Synchronizing Genetic Relaxation Oscillators by Inter-cell Signaling

  10. Our Inspiration • Garcia-Ojalvo, Elowitz et al. Modeling a synthetic multicellular clock: Repressilators coupled by quorum sensing

  11. The Goal! • The Oscillator Repressilator System J40001 I5610 I15004

  12. Simulations • Coupling the System to the Repressilator • Simulations and the Coupling Uncoupled Coupled

  13. Simulations Does the Quorum Sensor Oscillate?

  14. The Simulations • The Theoretical View • Mathematical Modeling • Deterministic Ordinary Differential Equations • Implemented using Mathematica xlr8r

  15. Simulations • Parameters: • Simulate Network of Cells which act in Synergy • Transcription • Translation • Coupling Constants • Reaction Coefficients • And the list goes on…

  16. Simulations • Uncoupled Example

  17. uncoupled coupled Simulations • Mathematica xlr8r • Network of Coupled Cells • Random Initial Conditions • Synchronization 1-2 periods • Predicted Oscillations

  18. Simulations • Continuum Approximations • Modeled from Elowitz System • Transform into Uncoupled, Linear System of Ordinary Differential Equations

  19. Simulations • Phase Portrait [Lac] [LuxI]

  20. Simulations • System Progression from any point • The Dynamics are Attracted towards the Fixed Point • Explains the Dampening of Oscillations… over time • Disturbances  from stochastic influences • Low plasmid copy number • Time Delay Constant (AI) • Phase Portrait [Lac] [LuxI]

  21. In the Lab • Team Awesome Oscillators • The Oscillator • Team Triple Ligation (Hai-Ya) • A new Construct • Theory Team • The simulators + Algorithm Construction

  22. In the Lab • Oscillating Components I15004: LuxR generator + LacI regulated lambda cI (LVA)+LuxI (LVA) J40001: LacI(LVA)+ECFP(LVA) under luxpR promoter(LuxR/HSL reg.)

  23. The Quorum Sensing System

  24. In the Lab • Simulations predicted Oscillations • Results were reproduced in the Lab • Coupled vs. Uncoupled

  25. Recreation • Recreation of Oscillating System • I + J  Zeiss510 Confocal Microscope

  26. In the Lab • Plate Reader  Spectrophotometer • Quantitative Assay • 96-well Reader • 17 hours; Pictures every 5 minutes • More Efficient • Effective usage of Lab Time/Resources

  27. Simulations • IPTG • Lac Analog • Simulation Predictions: • Addition of Lac blocks the system • Allows the system to be reset

  28. In the Lab • Plate Reader Experiment • IPTG • Dilute • Concentrated • Is this what we expected?

  29. Simulations • Dox • Tetracyclin Analog • Simulation Predictions: • Eliminate LuxR as a Limiting Factor • Uncouple the system

  30. In the Lab • Plate Reader Experiment • Dox • Dilute • Concentrated • Is this what we expected?

  31. Simulations • AHL • The AI • Simulation Predictions: • Stalls the System • Faster Synchronization

  32. In the Lab • Plate Reader Experiment • AHL • Dilute • Concentrated • Is this what we expected?

  33. The Control • J40001-Alone Experiments • Do the BL21 cells’ endogenous Lac affect oscillations? • Is the I15004 brick contributing? • Different Parameters • Expected from Simulations: No oscillations • Results: Well…

  34. Results • J40001 Alone Experiments • Alone • IPTG • Dox • Dilute • Concentrated

  35. Results • J40001 Alone Experiments • AHL • IPTG + Dox • IPTG + AHL • Dilute • Concentrated

  36. The Oscillations • Caveat… • Oscillations only in BL21 (lac+) • No Oscillations in MC4100 (lac-) BL21 Strain

  37. Results • Conclusions: • The I15004 Brick is sub-optimal • Does not give enough information • Want true Constitutive LuxR and a second Reporter Gene

  38. Construction • Similar brick to I15004 synthesized • pLac * rbs * luxI * RFP * stop * pConst * rbs * luxR * stop • Not yet Arrived… I724007!

  39. Construction • Addition of Aiia • Degradation of Autoinducer • Decrease half-life • Stabilize System • Prevent Flooding of AI

  40. Team Triple Ligation • B0034 + C0060  “B+C” • RBS aiiA • R0062 + “B+C”  “R+B+C” • pLux • “R+B+C” + J40001 • aiiA Lac generator

  41. Construction • New Constructs I724000! I724001!

  42. Original System • Coupling the System to the Repressilator • Stable, Sinusoidal Oscillations

  43. In the Lab • Dysfunctional Repressilator • Anomalous Gel Results • No Fluorescence • Is I5610 functional?

  44. Construction • Functional Repressilator • I5611 – LVA(-) • E0434 – YFP + LVA

  45. Construction BsrGI SpeI I5611 + YFP with LVA Cut I5611 Amp Cm Amp Ligate Cm BsrGI SpeI E0434 Amp I724006!

  46. Simulations • Future Directions • Stochastic Model; 3D System • Gillespie Algorithm • Diffusion • Transmembrane Transport • Continuous: Continuum Approximations • Discrete: Computationally Intensive & small number of molecules • Developing a new algorithm directly used with novel hardware

  47. Conclusions • Our system still has a lot of potential • E.Coli Mammals • Baby steps leading to applications in Medicine and Biotechnology • Plate Reader Experiments, Microscopy, Simulations, Fiddling with variables •  Lead to a better understanding of a very complicated system essential in Humans and Nature

  48. C'est Fini Merci!

  49. Thanks to everyone involved: Prof. Jay Nadeau Elvis Prandzic Adrian Kaats Hanmo Li Dongting Yu Stephanie Yee Alexandre David Allan Lim Jimmy Huynh Susan Westfall James Gabriels Ariq Emtenan Adam KatolikAlix Dudley Horia Vulpe Justin Ang Scott Gallagher Tim Johnstone Avi Biswas

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