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Quorum Sensing

Quorum Sensing. Stephanie Lo, 23 July 07. Review. Some Lessons Learned. Growth of bacteria only minimally affected by plate reader cover; much less drying out Condensation = problem for accuracy? Random wavelengths to measure growth did not work 360/420; 790/850. Biobricks. Senders.

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Quorum Sensing

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  1. Quorum Sensing Stephanie Lo, 23 July 07

  2. Review

  3. Some Lessons Learned • Growth of bacteria only minimally affected by plate reader cover; much less drying out • Condensation = problem for accuracy? • Random wavelengths to measure growth did not work • 360/420; 790/850

  4. Biobricks Senders Receiver S03623 I13507 T9002 I13507 S03608

  5. Experiment Details • Grew up liquid cultures of overnight sender (SI) and receiver (T02) • Dilution with LB to same OD • Innoculate with different dilutions of sender to receiver • Manually use plate reader to measure fluorescence (RFP and GFP) and OD • Shake plate at 37 degrees between reads

  6. Specifics • Experiment A and B were run manually, C was overnight • Initial ODs around 0.5 (Experiment A and C) and 0.25 (Experiment B)

  7. Correlation of RFP to Sender (SI) growth over small OD rangeWhat we expect to see RFP (RFU) OD

  8. RFP (RFU) versus OD Experiment A

  9. RFP versus time … Experiment A

  10. OD versus Time Experiment A

  11. GFP fluorescence vs time Experiment B

  12. RFP versus time Note the time lag until substantial RFP Experiment C

  13. Fluorescence vs. Timeovernight, 7/16 Initial OD = 0.055

  14. What we’d like to see – Quorum Activity GFP fluorescence per receiver (T02) cell OHHL concentration / Sender (SI) amount added / RFP fluorescence

  15. Detecting a Quorum System Against a Control GFP fluorescence per OD Quorum system Noninduced (actual) Noninduced (ideal) time

  16. GFP Fluorescence vs. Time Experiment A

  17. GFP versus Sender Added Experiment A

  18. GFP Fluorescence per Sender Normalized for Number of Receiver Cells Experiment B

  19. GFP per Receiver Cell at One Hour Experiment C

  20. Absolute GFP vs Time Experiment C

  21. GFP per receiver vs. time Experiment C

  22. Interestingly (and in contrast to JT system) …

  23. JT Induced versus Noninduced over time 7/16

  24. In the future: Characterize JT system as well? • Dilute samples to maintain relatively constant ODs / number of cells • Determine percent fluorescent through FACS or fluorescence microscope Percentage cells fluorescent OD

  25. Preliminary Experiences with Microscope

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