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

Quorum Sensing

Stephanie Lo, 23 July 07


Review

Review


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)


Correlation of rfp to sender si growth over small od range what we expect to see

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

RFP (RFU)

OD


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


Gfp fluorescence vs time

GFP fluorescence vs time

Experiment B


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 vs time1

GFP Fluorescence vs. Time

Experiment A


Gfp versus sender added

GFP versus Sender Added

Experiment A


Gfp fluorescence per sender normalized for number of receiver cells

GFP Fluorescence per Sender Normalized for Number of Receiver Cells

Experiment B


Gfp per receiver cell at one hour

GFP per Receiver Cell at One Hour

Experiment C


Absolute gfp vs time

Absolute GFP vs Time

Experiment C


Gfp per receiver vs time

GFP per receiver vs. time

Experiment C


Interestingly and in contrast to jt system

Interestingly (and in contrast to JT system) …


Jt induced versus noninduced over time

JT Induced versus Noninduced over time

7/16


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


Preliminary experiences with microscope

Preliminary Experiences with Microscope


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