Limits...
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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Model-basedexploration of parameter space. (A) Simulations ofscaffold to antiscaffold inputs and outputs over a range of phosphatase(100–5100 nM, 500 nM increments) and response regulator (10–1510nM, 150 nM increments) concentrations. Enlargement shows the scaffoldsingle occupancy limit concentration and curve fitting for each curve.Red dotted lines show curve fits; the slope represents the antiscaffoldto scaffold ratio. (B) Heat map showing antiscaffold to scaffold ratiofor each curve shown in part A. Increasing response regulator resultsin greater AS/Sc ratios. Gray box represents estimated experimentalphosphatase induction range. Black box estimates experimental responseregulator induction range. (C) Heat map of maximum scaffold occupancylimit. Higher concentrations of phosphatase result in decreased maximumscaffold occupancy limit.
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fig6: Model-basedexploration of parameter space. (A) Simulations ofscaffold to antiscaffold inputs and outputs over a range of phosphatase(100–5100 nM, 500 nM increments) and response regulator (10–1510nM, 150 nM increments) concentrations. Enlargement shows the scaffoldsingle occupancy limit concentration and curve fitting for each curve.Red dotted lines show curve fits; the slope represents the antiscaffoldto scaffold ratio. (B) Heat map showing antiscaffold to scaffold ratiofor each curve shown in part A. Increasing response regulator resultsin greater AS/Sc ratios. Gray box represents estimated experimentalphosphatase induction range. Black box estimates experimental responseregulator induction range. (C) Heat map of maximum scaffold occupancylimit. Higher concentrations of phosphatase result in decreased maximumscaffold occupancy limit.

Mentions: Circuitlimitations were explored in silico. Specifically,we investigated the effects of tuning response regulator and phosphataseconcentrations on the ability of the antiscaffold output to trackthe scaffold reference. Response regulator and phosphatase concentrationsare easily accessible parameters via inducible promoters in our experimentalsystem. In Figure 6A, a scan of input–outputresponse curves is shown over a range of response regulator and phosphataseconcentrations (See Figure S10, Supporting Information, for explicit values). For each curve in the grid, the scaffoldconcentration in which the single occupancy drop-off occurs was found,and the slope of the curve up to that concentration was found witha linear fit. The maximum scaffold occupancy limit is the concentrationof scaffold molecules at which each scaffold molecule only has eithera response regulator or histidine kinase. The slope of the curve upto that point represents the antiscaffold to scaffold ratio that canbe achieved by the circuit. In the case where the single occupancylimit does not appear, the last concentration is used. Data shownin Figure 6B indicates that increasing responseregulator values result in a greater AS/Sc ratio (up to 1.5-fold increase),while increasing phosphatase serves to bring down that ratio. Theeffect of increasing phosphatase is apparent when the maximum scaffoldoccupancy limit is examined (Figure 6C). Furthermore,the simulations show that some minimal amount of phosphatase is necessaryfor a sufficiently high response regulator turnover rate so as toapproach a 1:1 ratio. As phosphatase concentration increases, activeresponse regulators are quickly dephosphorylated, decreasing the efficacyof the scaffolds, lowering the maximum occupancy concentration, andmaking the drop-off more steep. Based on experimental outputs of ourcircuit, however, we believe the actual achievable dynamic range ofthe circuit is limited to the lower left corner of the parameter space.The qualitatively estimated induction range is shown with the blackand gray rectangles in Figure 6B,C.


Design and implementation of a biomolecular concentration tracker.

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

Model-basedexploration of parameter space. (A) Simulations ofscaffold to antiscaffold inputs and outputs over a range of phosphatase(100–5100 nM, 500 nM increments) and response regulator (10–1510nM, 150 nM increments) concentrations. Enlargement shows the scaffoldsingle occupancy limit concentration and curve fitting for each curve.Red dotted lines show curve fits; the slope represents the antiscaffoldto scaffold ratio. (B) Heat map showing antiscaffold to scaffold ratiofor each curve shown in part A. Increasing response regulator resultsin greater AS/Sc ratios. Gray box represents estimated experimentalphosphatase induction range. Black box estimates experimental responseregulator induction range. (C) Heat map of maximum scaffold occupancylimit. Higher concentrations of phosphatase result in decreased maximumscaffold occupancy limit.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Model-basedexploration of parameter space. (A) Simulations ofscaffold to antiscaffold inputs and outputs over a range of phosphatase(100–5100 nM, 500 nM increments) and response regulator (10–1510nM, 150 nM increments) concentrations. Enlargement shows the scaffoldsingle occupancy limit concentration and curve fitting for each curve.Red dotted lines show curve fits; the slope represents the antiscaffoldto scaffold ratio. (B) Heat map showing antiscaffold to scaffold ratiofor each curve shown in part A. Increasing response regulator resultsin greater AS/Sc ratios. Gray box represents estimated experimentalphosphatase induction range. Black box estimates experimental responseregulator induction range. (C) Heat map of maximum scaffold occupancylimit. Higher concentrations of phosphatase result in decreased maximumscaffold occupancy limit.
Mentions: Circuitlimitations were explored in silico. Specifically,we investigated the effects of tuning response regulator and phosphataseconcentrations on the ability of the antiscaffold output to trackthe scaffold reference. Response regulator and phosphatase concentrationsare easily accessible parameters via inducible promoters in our experimentalsystem. In Figure 6A, a scan of input–outputresponse curves is shown over a range of response regulator and phosphataseconcentrations (See Figure S10, Supporting Information, for explicit values). For each curve in the grid, the scaffoldconcentration in which the single occupancy drop-off occurs was found,and the slope of the curve up to that concentration was found witha linear fit. The maximum scaffold occupancy limit is the concentrationof scaffold molecules at which each scaffold molecule only has eithera response regulator or histidine kinase. The slope of the curve upto that point represents the antiscaffold to scaffold ratio that canbe achieved by the circuit. In the case where the single occupancylimit does not appear, the last concentration is used. Data shownin Figure 6B indicates that increasing responseregulator values result in a greater AS/Sc ratio (up to 1.5-fold increase),while increasing phosphatase serves to bring down that ratio. Theeffect of increasing phosphatase is apparent when the maximum scaffoldoccupancy limit is examined (Figure 6C). Furthermore,the simulations show that some minimal amount of phosphatase is necessaryfor a sufficiently high response regulator turnover rate so as toapproach a 1:1 ratio. As phosphatase concentration increases, activeresponse regulators are quickly dephosphorylated, decreasing the efficacyof the scaffolds, lowering the maximum occupancy concentration, andmaking the drop-off more steep. Based on experimental outputs of ourcircuit, however, we believe the actual achievable dynamic range ofthe circuit is limited to the lower left corner of the parameter space.The qualitatively estimated induction range is shown with the blackand gray rectangles in Figure 6B,C.

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