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Design and implementation of a biomolecular concentration tracker.

Hsiao V, de los Santos EL, Whitaker WR, Dueber JE, Murray RM - ACS Synth Biol (2014)

Bottom Line: Active feedback in closed loop systems offers a dynamic and adaptive way to ensure constant relative activity independent of intrinsic and extrinsic noise.In this work, we use synthetic protein scaffolds as a modular and tunable mechanism for concentration tracking through negative feedback.Input to the circuit initiates scaffold production, leading to colocalization of a two-component system and resulting in the production of an inhibitory antiscaffold protein.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, California United States.

ABSTRACT
As a field, synthetic biology strives to engineer increasingly complex artificial systems in living cells. Active feedback in closed loop systems offers a dynamic and adaptive way to ensure constant relative activity independent of intrinsic and extrinsic noise. In this work, we use synthetic protein scaffolds as a modular and tunable mechanism for concentration tracking through negative feedback. Input to the circuit initiates scaffold production, leading to colocalization of a two-component system and resulting in the production of an inhibitory antiscaffold protein. Using a combination of modeling and experimental work, we show that the biomolecular concentration tracker circuit achieves dynamic protein concentration tracking in Escherichia coli and that steady state outputs can be tuned.

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Multistep induction oftracker circuit. (A) Simulation resultsfor a three step induction show overlapping response times with eachcurve decreasing based on degradation rate after induction ceases.Upper panel shows aTc induction pattern with 1 h steps increasingin 50 nM increments starting 30 min after start of experiment. (B)Experimental time traces for Sc-RFP show overlapping fluorescenceoutput, with each curve decreasing at a time proportional to the numberof steps. Corresponding antiscaffold–YFP data show similaroverlaps and proportional decreases. Fluorescent measurements arenormalized such that the maximum value of the one step curve is 1au to better visualize fold change. Growth curves are shown in FigureS8, Supporting Information.
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fig4: Multistep induction oftracker circuit. (A) Simulation resultsfor a three step induction show overlapping response times with eachcurve decreasing based on degradation rate after induction ceases.Upper panel shows aTc induction pattern with 1 h steps increasingin 50 nM increments starting 30 min after start of experiment. (B)Experimental time traces for Sc-RFP show overlapping fluorescenceoutput, with each curve decreasing at a time proportional to the numberof steps. Corresponding antiscaffold–YFP data show similaroverlaps and proportional decreases. Fluorescent measurements arenormalized such that the maximum value of the one step curve is 1au to better visualize fold change. Growth curves are shown in FigureS8, Supporting Information.

Mentions: Followingstep input characterization, we investigated circuit response to multiplestep-up inputs. Figure 4 shows the resultsof a three step scaffold induction experiment with 1 h steps correspondingto 50 nM increases of aTc inducer. Growth curves are shown in FigureS8, Supporting Information. The single negativefeedback loop in the circuit represses overproduction of antiscaffold,but there is no mechanism for feedback in the case of an excess ofscaffold or antiscaffold. As such, the model predicts that increasesin inducer will lead to immediate increases of scaffold followed closelyby the antiscaffold but once induction is turned off, degradationof proteins depends on the endogenous ClpXP degradation machinery(Figure 4A). Additionally, the upward slopeof each curve should overlap until induction ceases.


Design and implementation of a biomolecular concentration tracker.

Hsiao V, de los Santos EL, Whitaker WR, Dueber JE, Murray RM - ACS Synth Biol (2014)

Multistep induction oftracker circuit. (A) Simulation resultsfor a three step induction show overlapping response times with eachcurve decreasing based on degradation rate after induction ceases.Upper panel shows aTc induction pattern with 1 h steps increasingin 50 nM increments starting 30 min after start of experiment. (B)Experimental time traces for Sc-RFP show overlapping fluorescenceoutput, with each curve decreasing at a time proportional to the numberof steps. Corresponding antiscaffold–YFP data show similaroverlaps and proportional decreases. Fluorescent measurements arenormalized such that the maximum value of the one step curve is 1au to better visualize fold change. Growth curves are shown in FigureS8, Supporting Information.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4384833&req=5

fig4: Multistep induction oftracker circuit. (A) Simulation resultsfor a three step induction show overlapping response times with eachcurve decreasing based on degradation rate after induction ceases.Upper panel shows aTc induction pattern with 1 h steps increasingin 50 nM increments starting 30 min after start of experiment. (B)Experimental time traces for Sc-RFP show overlapping fluorescenceoutput, with each curve decreasing at a time proportional to the numberof steps. Corresponding antiscaffold–YFP data show similaroverlaps and proportional decreases. Fluorescent measurements arenormalized such that the maximum value of the one step curve is 1au to better visualize fold change. Growth curves are shown in FigureS8, Supporting Information.
Mentions: Followingstep input characterization, we investigated circuit response to multiplestep-up inputs. Figure 4 shows the resultsof a three step scaffold induction experiment with 1 h steps correspondingto 50 nM increases of aTc inducer. Growth curves are shown in FigureS8, Supporting Information. The single negativefeedback loop in the circuit represses overproduction of antiscaffold,but there is no mechanism for feedback in the case of an excess ofscaffold or antiscaffold. As such, the model predicts that increasesin inducer will lead to immediate increases of scaffold followed closelyby the antiscaffold but once induction is turned off, degradationof proteins depends on the endogenous ClpXP degradation machinery(Figure 4A). Additionally, the upward slopeof each curve should overlap until induction ceases.

Bottom Line: Active feedback in closed loop systems offers a dynamic and adaptive way to ensure constant relative activity independent of intrinsic and extrinsic noise.In this work, we use synthetic protein scaffolds as a modular and tunable mechanism for concentration tracking through negative feedback.Input to the circuit initiates scaffold production, leading to colocalization of a two-component system and resulting in the production of an inhibitory antiscaffold protein.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, California United States.

ABSTRACT
As a field, synthetic biology strives to engineer increasingly complex artificial systems in living cells. Active feedback in closed loop systems offers a dynamic and adaptive way to ensure constant relative activity independent of intrinsic and extrinsic noise. In this work, we use synthetic protein scaffolds as a modular and tunable mechanism for concentration tracking through negative feedback. Input to the circuit initiates scaffold production, leading to colocalization of a two-component system and resulting in the production of an inhibitory antiscaffold protein. Using a combination of modeling and experimental work, we show that the biomolecular concentration tracker circuit achieves dynamic protein concentration tracking in Escherichia coli and that steady state outputs can be tuned.

Show MeSH
Related in: MedlinePlus