Part:BBa_K4294803
PFc
Usage and Biology
This is a circuit of the LuxR quorum sensing regulator in a positive feedback loop with an 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.
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Figure 1: Flow chart of PFR (Positive Feedback + Repressor) circuit. This topology resembles the one of a coherent feedforward loop with an added positive involving the first step.
Figure 1: PFR genetic circuit overview. The output is controlled by the hybrid promoter BBa_K4294301, which is activated by LuxR and repressed by PhlF. LuxR is placed downstream of the Plux promoter, establishing a positive feedback after induction with OC6 as previously described. LuxR also binds to a Plux repressible promoter (BBa_J107103), which controls the production of PhlF, blocking PhlF expression after induction with OC6. Every coding sequence is placed downstream a BBa_B0034 RBS. PhlF has a high efficiency NDENYALAA degradation tag.
PFR circuit at its “OFF” state (“0”)
PFR circuit 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 1: Induction of BL21 PFr pTU2-RFP colE1 or. with the following OC6 concentrations (μΜ):100, 20, 4, 0.16, 0.0064, 0.00128, 0.000256, 0.00001024. 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2047
Illegal NheI site found at 2070 - 21INCOMPATIBLE WITH RFC[21]Illegal suffix found in sequence at 970
Illegal BglII site found at 958
Illegal BglII site found at 1978
Illegal BamHI site found at 1990
Illegal XhoI site found at 1999 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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