Part:BBa_K4294802
PF
This is a circuit of the LuxR quorum sensing regulator in a positive feedback loop.
Usage and Biology
This induction system is a quorum sensing system derived from Vibrio fisheri [1] It is generally used in synthetic biology to programming communication between bacterial populations and is well characterized in the bibliography [2] A positive feedback loop is a very common mode of action in different biological systems.
Circuit Design
One already tested and widely utilized method to engineer a bistable circuit, is transcriptional Positive Feedbacks. In such circuits, an activator of an output induces its own production as well after induction with its respective ligand.
In our case, Plux was placed upstream of the LuxR coding sequence and the output coding sequence. Therefore, LuxR activates its own production after binding with OC6 and establishes positive feedback.
In this case, LuxR is being controlled only by the Plux promoter.
Plux leakiness provides a low basal LuxR concentration in the cell. This concentration is sufficient for the LuxR dimer formation after induction with OC6. The activated LuxR dimers promote the expression more LuxR resulting in positive feedback.
When the concentration of OC6 increases enough, it binds with LuxR, LuxR forms stable dimmers and binds to its binding site (lux box), upstream of the -35 region of the Plux promoter, activating the transcription of the downstream output.
PF (Positive Feedback) genetic circuit overview. LuxR is placed downstream of the promoter Plux inducible promoter and BBa_B0034 RBS. The leakiness of Plux promoter provides a basal LuxR concentration in the cell that will be activated by OC6 and promote the production of more LuxR and of the output, resulting in a positive feedback loop. Output (mNeonGreen) is placed downstream Plux promoter and BBa_B0034 RBS.
PF at is “OFF” state (‘“0”) PF at its “ON” state (“1”)
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Figure 1: Flowchart of a positive feedback circuit.
Figure 2: PF (Positive Feedback) genetic circuit overview. LuxR is placed downstream of the promoter Plux inducible promoter and BBa_B0034 RBS. The leakiness of Plux promoter provides a basal LuxR concentration in the cell that will be activated by OC6 and promote the production of more LuxR and of the output, resulting in a positive feedback loop. Output (mNeonGreen) is placed downstream Plux promoter and BBa_B0034 RBS.
Figure 3: PF at is “OFF” state (‘“0”).
Figure 4: PF at is “ON” state (‘“1”)
Measurment
In our design, the downstream output was mNeonGreen fluorescent protein. To quantify this output we measured the fluorescence using microplate reader FlexStation3 (Molecular Devices) with an excitation wavelength 476nm and emission wavelength 547nm as suggested by the manufacturer. We conducted measurements at different time points after the induction with OC6, using different concentrations of the inducer.
Figure 5: Induction of BL21 PF pTU2-RFP colE1 or. with the following OC6 concentrations (μΜ): 0.062, 0.031, 0.0155, 0.003875, 0.0019375, 0.00096875, 0.000484375, 0.000242188, 0.000121094. The uninduced cells are represented by the concentration value 0.00001 for the diagram purposes.
References
[1] Dunlap P. V. (1999). Quorum regulation of luminescence in Vibrio fischeri. Journal of molecular microbiology and biotechnology, 1(1), 5–12.
[2] R.M. Davis,Front. Bioeng. Biotechnol., 10 March 2015 Sec. Synthetic Biology https://doi.org/10.3389/fbioe.2015.00030
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal suffix found in sequence at 928
Illegal BglII site found at 916 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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