Quorum Sensing: Week 6 Results

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Quorum Sensing Week 6 Results

• Two Cell vs. One Cell Systems

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Switched Fluorophores
Original Constructs
Senders
lacI regulated RFP Sender (S08I07)
tetR regulated RFP Sender (S23I07)
Receiver
GFP Receiver (T02)
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Switched Fluorophores
Flipped Constructs
Senders
lacI regulated GFP Sender (S08E40)
tetR regulated GFP Sender (S23E40)
Receiver
RFP Receiver (F20I07)
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Switched Fluorophores
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Switched Fluorophores
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Switched Fluorophores

• Protein Folding/Maturation issue?
• GFP (E0040)
• gfpmut3b
• The expression of GFP is observable minutes
  after induction1
• RFP (E1010)
• mRFP1 - highly engineered mutant of red
  fluorescent protein from Discosoma striata
  (coral)
• Based on our data at 37C, we estimate the t0.5
  for maturation of mRFP1 and T1 is less than 1 h,
  consistent with the 0.7 h previously reported for
  T12

1 FACS-optimized mutants of the green
fluorescent protein (GFP).Gene 0378-1119
Cormack yr1996 vol173 iss1 Spec No pg33 -8 2
A monomeric red fluorescent protein. Proc Natl
Acad Sci U S A. 2002 June 11 99(12) 78777882.
doi 10.1073/pnas.082243699.
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FACS - Inspiration
Anderson, J. C., Clarke, E. J., Arkin, A. P.,
Voigt, C. A. (2006) Environmentally Controlled
Invasion of Cancer Cells by Engineered Bacteria,
Journal of Molecular Biology, 355 (4) 619-627.
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FACS - Results
110 Dilution Final OD 0.02 Mean GFP 370
1100 Dilution Final OD 0.141 Mean GFP 315
11000 Dilution Final OD 0.555 Mean GFP 426