Quorum Sensing
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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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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

Receiver

S03623

I13507

T9002

I13507

S03608


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


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)


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

RFP (RFU)

OD


RFP (RFU) versus OD

Experiment A


RFP versus time …

Experiment A


OD versus Time

Experiment A


GFP fluorescence vs time

Experiment B


RFP versus time

Note the time lag until substantial RFP

Experiment C


Fluorescence vs. Timeovernight, 7/16

Initial OD = 0.055


What we’d like to see – Quorum Activity

GFP fluorescence per receiver (T02) cell

OHHL concentration /

Sender (SI) amount added /

RFP fluorescence


Detecting a Quorum System Against a Control

GFP fluorescence per OD

Quorum system

Noninduced (actual)

Noninduced (ideal)

time


GFP Fluorescence vs. Time

Experiment A


GFP versus Sender Added

Experiment A


GFP Fluorescence per Sender Normalized for Number of Receiver Cells

Experiment B


GFP per Receiver Cell at One Hour

Experiment C


Absolute GFP vs Time

Experiment C


GFP per receiver vs. time

Experiment C


Interestingly (and in contrast to JT system) …


JT Induced versus Noninduced over time

7/16


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


Preliminary Experiences with Microscope


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