Difference between revisions of "Part:BBa K4294803"

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PFc

Usage and Biology

This is a circuit of the LuxR quorum sensing regulator in a positive feedback loop with additional constitutive production of LuxR repressor.

Circuit Design

One already tested and widely utilized method to engineer a bistable circuit, are transcriptional Positive Feedbacks. In such circuits, an activator of an output induces its own production as well after induction with its respective ligand. A similar circuit was built by Li et. al [1] to achieve a steeper activation function of the CinR quorum sensing activator system.

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 we are providing an extra TU for constitutive LuxR production. This topology provides a higher basal LuxR concentration inside the cell. This circuit was constructed and tested in order to find the most suitable regulator and circuit dynamics for our system.

Figure 1: Flowchart of a positive feedback circuit.

Figure 2: PFc (Positive Feedback with constitutive LuxR production) genetic circuit overview. Two LuxR TU exist in this circuit. One expresses LuxR constitutively and provides a higher LuxR concentration in the cell. The other LuxR TU (with the Plux promoter) is responsible for the generation of the positive feedback after induction with OC6. Output (mNeonGreen) is placed downstream of the Plux promoter and BBa_B0034 RBS.

Figure 3: PFc at its “OFF” state (“0”)

Figure 3: PF at its “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 from the manufacturer. We conducted measurements in different time points after the induction with OC6, using different concentrations of the inducer.

Figure 5: Induction of BL21 PFc pTU2-RFP colE1 or. with the following OC6 concentrations (μΜ): 10, 10, 1, 0.1, 0.01, 0.001, 0.0001. The uninduced cells are represented by the concentration value 0.000001 for the diagram purposes.

References

[1] Li X, Rizik L, Kravchik V, Khoury M, Korin N, Daniel R. Synthetic neural-like computing in microbial consortia for pattern recognition. Nat Commun. 2021 May 25;12(1):3139. doi: 10.1038/s41467-021-23336-0. PMID: 34035266; PMCID: PMC8149857.

Sequence and Features