Difference between revisions of "Part:BBa K4294812"
 
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<partinfo>BBa_K4294812 short</partinfo>
 
<partinfo>BBa_K4294812 short</partinfo>
  
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This part is a transcriptional unit capable to produce mNeonGreen as output when LuxR binds with its operator.
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==Design==
  
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Its' function for our project was to get induced by LuxR in order to proceed with the binary classification of a pattern, according to the amount of an intermediate molecule (OC6) produced by the senders' population.
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For transcriptionally regulated gene expression, one approach to achieve a switch-like response is an engineered promoter onto which multiple transcription regulators bind cooperatively or a biochemical mimic of this effect.
  
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==Design==
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This led us to the design of 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.
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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].
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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.
  
 
We used this part for our Positive Feedback Repressive construct (BBa_K4294804) as well as for our Positive Feedback with constitutive production of LuxR - repressive construct (BBa_K4294805).
 
We used this part for our Positive Feedback Repressive construct (BBa_K4294804) as well as for our Positive Feedback with constitutive production of LuxR - repressive construct (BBa_K4294805).
  
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<!-- Add more about the biology of this part here
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==References==
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===Usage and Biology===
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[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.
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[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.
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[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.
  
 
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Latest revision as of 15:28, 12 October 2022


TU Output for PF+ R, PFc+ R

Design

For transcriptionally regulated gene expression, one approach to achieve a switch-like response is an engineered promoter onto which multiple transcription regulators bind cooperatively or a biochemical mimic of this effect.

This led us to the design of 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.

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.

We used this part for our Positive Feedback Repressive construct (BBa_K4294804) as well as for our Positive Feedback with constitutive production of LuxR - repressive construct (BBa_K4294805).

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
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]