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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

Stephanie Lo, 23 July 07



Some lessons learned
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
Biobricks

Senders

Receiver

S03623

I13507

T9002

I13507

S03608


Experiment details
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
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)



Rfp rfu versus od
RFP (RFU) versus OD

Experiment A


Rfp versus time
RFP versus time …

Experiment A


Od versus time
OD versus Time

Experiment A



Rfp versus time1
RFP versus time

Note the time lag until substantial RFP

Experiment C


Fluorescence vs time overnight 7 16
Fluorescence vs. Timeovernight, 7/16

Initial OD = 0.055


What we d like to see quorum activity
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
Detecting a Quorum System Against a Control

GFP fluorescence per OD

Quorum system

Noninduced (actual)

Noninduced (ideal)

time




Gfp fluorescence per sender normalized for number of receiver cells
GFP Fluorescence per Sender Normalized for Number of Receiver Cells

Experiment B



Absolute gfp vs time
Absolute GFP vs Time

Experiment C





In the future characterize jt system as well
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



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