Quorum Sensing

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Quorum Sensing

• Stephanie Lo
• Harvard iGEM
• 8/6/07

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(No Transcript)
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After Overnight at 37C
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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

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JT Assay
Initial Diluted Sample OD 0.3 Fluorescence 600
Saturated Overnight Culture OD 1.5 Fluorescence
3000
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JT Assay
Initial Diluted Sample OD 0.3 Fluorescence 600
Growth OD 0.3 Fluorescence lt 600
Second (immediate) dilution OD 0.1 Fluorescence
200
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Quorum/growth
dilution
Fluorescence
OD
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Expected Results Fluorescence per OD
Fluorescence / OD
After overnight growth
Before 1st run
Saturated overnight
After 1st run
After 2nd run
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Characterizing JT 7/31
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Characterizing JT 7/31
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Characterizing JT 7/31
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Characterizing JT 7/31
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Characterizing JT 7/31
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Characterizing JT 8/1
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Fluorescence results as expected, growth curve a
concern
Characterizing JT 8/1
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New Approach?
Characterizing JT 8/2

• 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

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Plate Reader Endpoints 3rd Attempt
Characterizing JT 8/3
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Unexpectedly scarce absolute fluorescence growth
Characterizing JT 8/3
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Characterizing JT 8/3
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Characterizing JT 8/3
Is this an issue involving lack of growth?
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ODs after overnight indicate saturation
Characterizing JT 8/3
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Characterizing Sender/Receiver Systems
CELLS
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Characterizing Sender/Receiver Systems
Expected Results
Above quorum
Absolute Fluorescence or Fluorescence / OD
Below quorum
Amount supernatant (OHHL) added
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Growth Curves T02 induced
Characterizing Sender/Receiver Systems 8/1
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Characterizing Sender/Receiver Systems 8/1
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Characterizing Sender/Receiver Systems 8/1
Amount of SI added
Amount of SI added
Not shown JT Fl/OD after overnight 3630
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Better Characterization of Sender/Receiver System

• More samples with different amounts of
  supernatant added
• Different types of supernatant RACE, SE

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Characterizing Sender/Receiver Systems 8/3
Amount supernatant added
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Fluorescence over OD after Overnight
Characterizing Sender/Receiver Systems 8/3
Expected trend, wrong magnitude?
Amount supernatant added
Not shown Nondiluted JT 990 RFU/OD
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Increase in fluorescence is not drastic
Characterizing Sender/Receiver Systems 8/3
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Fluorescence over OD gives puzzling results
Characterizing Sender/Receiver Systems 8/3