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Quorum Sensing Week 6 Results: Evaluation of Two-Cell vs One-Cell Systems with Fluorophores

This report presents the results from Week 6 of our Quorum Sensing project, focusing on the performance of two-cell versus one-cell systems using various fluorophores. We constructed and evaluated lacI-regulated RFP and tetR-regulated RFP senders alongside their GFP counterparts. The expression and maturation rates of the fluorophores, including GFP and mRFP1, were analyzed, highlighting observable expression changes soon after induction. Key findings on dilution effects and the importance of protein folding/maturation are discussed, providing insights into engineered fluorescent protein behaviors.

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Quorum Sensing Week 6 Results: Evaluation of Two-Cell vs One-Cell Systems with Fluorophores

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  1. Quorum Sensing: Week 6 Results Two Cell vs. One Cell Systems

  2. Switched Fluorophores Original Constructs Senders lacI regulated RFP Sender (S08I07) tetR regulated RFP Sender (S23I07) Receiver GFP Receiver (T02)

  3. Switched Fluorophores Flipped Constructs Senders lacI regulated GFP Sender (S08E40) tetR regulated GFP Sender (S23E40) Receiver RFP Receiver (F20I07)

  4. Switched Fluorophores

  5. Switched Fluorophores

  6. Switched Fluorophores • Protein Folding/Maturation issue? • GFP (E0040) • gfpmut3b • “The expression of GFP is observable minutes after induction”1 • RFP (E1010) • mRFP1 - “**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)” • “Based on our data at 37°C, 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 T1”2 1 “FACS-optimized mutants of the green fluorescent protein (GFP).”Gene [0378-1119] Cormack yr:1996 vol:173 iss:1 Spec No pg:33 -8 2 “A monomeric red fluorescent protein.” Proc Natl Acad Sci U S A. 2002 June 11; 99(12): 7877–7882. doi: 10.1073/pnas.082243699.

  7. 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.

  8. FACS - Results 1:10 Dilution Final OD = 0.02 Mean GFP = 370 1:100 Dilution Final OD = 0.141 Mean GFP = 315 1:1000 Dilution Final OD = 0.555 Mean GFP = 426

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