Part:BBa_K4294420
PhlF with a degradation tag
Design to create a steep activation function
PhlF was chosen for our constructs 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 [1]).
We used it for our PFR (BBa_K4294804) and PFc+R (BBa_K4294805) receivers' constructs for transcriptionally regulated gene expression. Combine it with LuxR system we design also 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.
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 1: LuxBox and PhlO operator Hybrid Promoter
Figure 2: PFc+R receivers' circuit
Figure 3: PFR receivers' circuit
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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