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Quorum Sensing

Quorum Sensing. Stephanie Lo Harvard iGEM 8/6/07. After Overnight at 37C. Goals. Characterize JT (sender/receiver construct in one) Is quorum-like activity occurring? If so, at what OD is it detectable? Optimize ratio of separate sender-receiver cells. JT Assay. Initial Diluted Sample

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Quorum Sensing

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  1. Quorum Sensing Stephanie Lo Harvard iGEM 8/6/07

  2. After Overnight at 37C

  3. Goals • Characterize JT (sender/receiver construct in one) • Is quorum-like activity occurring? • If so, at what OD is it detectable? • Optimize ratio of separate sender-receiver cells

  4. JT Assay Initial Diluted Sample OD = 0.3 Fluorescence = 600 Saturated Overnight Culture OD = 1.5 Fluorescence = 3000

  5. JT Assay Initial Diluted Sample OD = 0.3 Fluorescence = 600 Growth OD = 0.3 Fluorescence < 600 Second (immediate) dilution OD = 0.1 Fluorescence = 200

  6. Quorum/growth dilution Fluorescence OD

  7. Expected Results: Fluorescence per OD Fluorescence / OD After overnight growth Saturated overnight Before 1st run After 1st run After 2nd run

  8. Characterizing JT 7/31

  9. Characterizing JT 7/31

  10. Characterizing JT 7/31

  11. Characterizing JT 7/31

  12. Characterizing JT 7/31

  13. Characterizing JT 8/1

  14. Characterizing JT 8/1 Fluorescence results as expected, growth curve a concern

  15. Characterizing JT 8/2 New Approach? • Past attempts: • Plate reader stopped on regular basis, endpoint fluorescence and OD read • New approach: • Split initial dilution in two • Leave plate reader on kinetic fluorescence • Manually read OD regularly at photospectrometer

  16. Characterizing JT 8/3 Plate Reader Endpoints: 3rd Attempt

  17. Characterizing JT 8/3 Unexpectedly scarce absolute fluorescence growth

  18. Characterizing JT 8/3

  19. Characterizing JT 8/3 Is this an issue involving lack of growth?

  20. Characterizing JT 8/3 ODs after overnight indicate saturation

  21. Characterizing Sender/Receiver Systems Supernatant LB CELLS

  22. Characterizing Sender/Receiver Systems Expected Results Above quorum Absolute Fluorescence or Fluorescence / OD Below quorum Amount supernatant (OHHL) added

  23. Characterizing Sender/Receiver Systems 8/1 Growth Curves: T02 induced

  24. Characterizing Sender/Receiver Systems 8/1

  25. Characterizing Sender/Receiver Systems 8/1 Amount of SI added Amount of SI added Not shown: JT Fl/OD after overnight = 3630

  26. Better Characterization of Sender/Receiver System • More samples with different amounts of supernatant added • Different types of supernatant: RACE, SE

  27. Characterizing Sender/Receiver Systems 8/3 Amount supernatant added

  28. Characterizing Sender/Receiver Systems 8/3 Fluorescence over OD after Overnight Expected trend, wrong magnitude? Amount supernatant added Not shown: Nondiluted JT: 990 RFU/OD

  29. Characterizing Sender/Receiver Systems 8/3 Increase in fluorescence is not drastic

  30. Characterizing Sender/Receiver Systems 8/3 Fluorescence over OD gives puzzling results

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