Part:BBa_K4294805

PFc+R

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

PhlF is under the transcriptional control of a LuxR repressible promoter (Plux_rep, BBa_J107103). However, in this promoter the LuxR binding domain is not placed upstream of the -35 hexamer, but inside the core sequence between the -35 and -10 regions. Due to this localization change, the effect of the active LuxR dimers is reversed; instead of activating the production of PhlF, they repress it [1].

Even though LuxR and PhlF do not bind cooperatively to their respective binding sites, their dynamics are indirectly connected. PhlF was chosen since it is an effective repressor with a low association constant K, indicating its ability to form functional dimers in lower concentrations (according to the Ecoli Marionette strains characterization [2]). Therefore, in “intermediate” OC6 concentrations, it could possibly still reinforce repression to the output irrespective of its lower production rate (due to the effect of LuxR on the Plux_rep promoter) and the activation effect of LuxR to the output’s promoter. LuxR establishes a positive feedback loop to propagate its effect and both positive feedback topologies (with and without constitutive LuxR expression) were designed and tested. A very efficient ssrA [3] degradation tag (NDENYALAA) was added to PhlF to ensure its rapid degradation after maximum repression of the phlF gene by LuxR and to avoid PhlF concentrations that might lead to a complete circuit block in every inducer concentration. The degradation tag was empirically selected and further analysis of different degradation tags will be needed for a potential optimization of the circuit’s response.

Figure 2: Flow chart of PFc+R (Positive Feedback with constitutive LuxR production + Repressor) circuits. This topology resembles the one of a coherent feedforward loop with an added positive involving the first step.

Figure 3: PFcR genetic circuit overview. This circuit is similar to the PFR circuit, but with an added constitutive expression of LuxR, which provides a higher basal LuxR concentration in the cell.

Figure 4: PFcR at is “OFF” state (‘“0”)

Figure 5: PFcR 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 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 6: Induction of BL21 PFc+r pTU2-RFP colE1 or. with the following OC6 concentrations (μΜ): 10, 2, 0.4, 0.08, 0.016, 0.0032, 0.00064, 0.000128, 0.0000256, 0.00000512. The uninduced cells are represented by the concentration value 0.0000001 for the diagram purposes.

References

[1] Zucca S, Pasotti L, Politi N, Casanova M, Mazzini G, Cusella De Angelis MG, Magni P. Multi-Faceted Characterization of a Novel LuxR-Repressible Promoter Library for Escherichia coli. PLoS One. 2015 May 26;10(5):e0126264. doi: 10.1371/journal.pone.0126264. PMID: 26010244; PMCID: PMC4444344.

[2] Meyer AJ, Segall-Shapiro TH, Glassey E, Zhang J, Voigt CA. Escherichia coli "Marionette" strains with 12 highly optimized small-molecule sensors. Nat Chem Biol. 2019 Feb;15(2):196-204. doi: 10.1038/s41589-018-0168-3. Epub 2018 Nov 26. PMID: 30478458.

[3] Fei X, Bell TA, Barkow SR, Baker TA, Sauer RT. Structural basis of ClpXP recognition and unfolding of ssrA-tagged substrates. Elife. 2020 Oct 22;9:e61496. doi: 10.7554/eLife.61496. PMID: 33089779; PMCID: PMC7652416.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 31
  Illegal NheI site found at 2883
  Illegal NheI site found at 2906
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal suffix found in sequence at 828
  Illegal BglII site found at 816
  Illegal BglII site found at 1794
  Illegal BglII site found at 2814
  Illegal BglII site found at 3814
  Illegal BamHI site found at 1806
  Illegal BamHI site found at 2826
  Illegal BamHI site found at 3826
  Illegal XhoI site found at 1815
  Illegal XhoI site found at 2835
  Illegal XhoI site found at 3835
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]