Part:BBa_K4294804

PFR

Here we use the part BBa_K4294301, a hybrid promoter which includes a LuxR activator binding domain (luxbox) upstream the -35 hexamer and a PhlF operator (phlO) that spans from the core sequence and the -10 hexamer to the proximal promoter sequence.

Figure 1: Plux-phlO hybrid promoter.

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.

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.

Due to wiki freeze related lagging, the composite part part connections are the following:

BBa_J107103 [0] BBa_B0034 [0] BBa_K4294420 [0] BBa_B0015 [0] BBa_K4294999 [cgta] BBa_K4294301 [0] BBa_B0034 [0] BBa_K4294410 [0] BBa_B0015 [0] BBa_K4294999 [gtca] BBa_R0062 [0] BBa_B0034 [0] BBa_K4294401 [0] BBa_B0015 [ttag]

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] Zucca, S., Pasotti, L., Politi, N., Casanova, M., Mazzini, G., Cusella De Angelis, M. G., & Magni, P. (2015). Multi-Faceted Characterization of a Novel LuxR-Repressible Promoter Library for Escherichia coli. PloS one, 10(5), e0126264. https://doi.org/10.1371/journal.pone.0126264

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2047
  Illegal NheI site found at 2070
  
• 21
  INCOMPATIBLE 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
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]