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Synthetic conversion of a graded receptor signal into a tunable, reversible switch.

Palani S, Sarkar CA - Mol. Syst. Biol. (2011)

Bottom Line: However, synthetic gene network 'switches' have been limited in their applicability and tunability due to their reliance on specific components to function.Here, we present a strategy for reversible switch design that instead relies only on a robust, easily constructed network topology with two positive feedback loops and we apply the method to create highly ultrasensitive (n(H)>20), bistable cellular responses to a synthetic ligand/receptor complex.Independent modulation of the two feedback strengths enables rational tuning and some decoupling of steady-state (ultrasensitivity, signal amplitude, switching threshold, and bistability) and kinetic (rates of system activation and deactivation) response properties.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104-6321, USA.

ABSTRACT
The ability to engineer an all-or-none cellular response to a given signaling ligand is important in applications ranging from biosensing to tissue engineering. However, synthetic gene network 'switches' have been limited in their applicability and tunability due to their reliance on specific components to function. Here, we present a strategy for reversible switch design that instead relies only on a robust, easily constructed network topology with two positive feedback loops and we apply the method to create highly ultrasensitive (n(H)>20), bistable cellular responses to a synthetic ligand/receptor complex. Independent modulation of the two feedback strengths enables rational tuning and some decoupling of steady-state (ultrasensitivity, signal amplitude, switching threshold, and bistability) and kinetic (rates of system activation and deactivation) response properties. Our integrated computational and synthetic biology approach elucidates design rules for building cellular switches with desired properties, which may be of utility in engineering signal-transduction pathways.

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Related in: MedlinePlus

Feedback modulation of kinetic and steady-state system responses. (A) Activation kinetics for cTF strains. Strains cRcTF, sRcTF, and tRcTF were induced with 1 μM IP and aliquots were taken from the cultures at different time points to determine GFP expression kinetics and steady-state levels. As these strains are all transcription factor limited, the increased receptor feedback does not enhance steady-state GFP expression. (B) Activation kinetics for tTF strains. Similarly, GFP expression kinetics and steady-state levels were quantified for strains cRtTF, sRtTF, and tRtTF after induction with 1 μM IP. The strong transcription factor feedback loop makes these strains receptor limited, so receptor feedback now has a significant effect on steady-state GFP expression. (C) Temporal activation profiles of all six strains normalized to their steady-state setpoint. All six strains reach 50% of the steady-state level at different times (Act50), with stronger feedback loops slowing the kinetics. (D) Temporal deactivation profiles of the strains normalized to the steady-state setpoint. After reaching steady-state GFP expression in 1 μM IP, the cultures were thoroughly washed and resuspended in media with no IP. All strains exhibit reversibility in the absence of IP, but the deactivation kinetics are markedly different. The time at which 50% of the initial GFP expression level is reached (Deact50) is fastest for the basic and receptor feedback strains and slowest for the double feedback strains. (E) Steady-state dose–response curves for cTF strains. For cRcTF, sRcTF, and tRcTF strains, the steady-state GFP responses to IP (0.01–10 μM) are indistinguishable and weakly ultrasensitive (nH∼2). When the strains were allowed to reach high GFP steady-state levels in 1 μM IP and were then reduced to sub-threshold concentrations of IP (0.012, 0.025, and 0.05 μM IP), the strains exhibited no memory and showed a monostable response (for clarity, reverse curves are not shown as they overlay the forward curves). (F) Steady-state dose–response curves for tTF strains. While the steady-state response for the cRtTF strain exhibits slightly greater ultrasensitivity (nH∼4), the dual-feedback strains sRtTF and tRtTF strikingly function as almost as pure binary switches (nH∼20). When the strains were allowed to reached high GFP steady-state levels in 1 μM IP and were then reduced to sub-threshold concentrations of IP (0.012, 0.025, and 0.05 μM IP), all three strains exhibited memory (reverse curves shown as dotted lines). (G) Deactivation kinetics for cRcTF strain. After reaching high GFP steady-state levels in 1 μM IP, strain cRcTF was thoroughly washed and resuspended in medium with different sub-threshold IP concentrations (0.012, 0.025, and 0.05 μM). The temporal deactivation curves indicate no temporal memory in this strain when compared with the 0 μM IP concentration curve (dotted gray line). (H) Deactivation kinetics for tRtTF strain. After reaching high GFP steady-state levels in 1 μM IP, strain tRtTF was thoroughly washed and resuspended in medium with different sub-threshold IP concentrations (0.012, 0.025, and 0.05 μM). For all sub-threshold concentrations, the strain shows temporal memory when compared with the 0 μM IP concentration curve (dotted gray line); however, the strain is only bistable at 0.05 μM IP. Source data is available for this figure at www.nature.com/msb.
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f2: Feedback modulation of kinetic and steady-state system responses. (A) Activation kinetics for cTF strains. Strains cRcTF, sRcTF, and tRcTF were induced with 1 μM IP and aliquots were taken from the cultures at different time points to determine GFP expression kinetics and steady-state levels. As these strains are all transcription factor limited, the increased receptor feedback does not enhance steady-state GFP expression. (B) Activation kinetics for tTF strains. Similarly, GFP expression kinetics and steady-state levels were quantified for strains cRtTF, sRtTF, and tRtTF after induction with 1 μM IP. The strong transcription factor feedback loop makes these strains receptor limited, so receptor feedback now has a significant effect on steady-state GFP expression. (C) Temporal activation profiles of all six strains normalized to their steady-state setpoint. All six strains reach 50% of the steady-state level at different times (Act50), with stronger feedback loops slowing the kinetics. (D) Temporal deactivation profiles of the strains normalized to the steady-state setpoint. After reaching steady-state GFP expression in 1 μM IP, the cultures were thoroughly washed and resuspended in media with no IP. All strains exhibit reversibility in the absence of IP, but the deactivation kinetics are markedly different. The time at which 50% of the initial GFP expression level is reached (Deact50) is fastest for the basic and receptor feedback strains and slowest for the double feedback strains. (E) Steady-state dose–response curves for cTF strains. For cRcTF, sRcTF, and tRcTF strains, the steady-state GFP responses to IP (0.01–10 μM) are indistinguishable and weakly ultrasensitive (nH∼2). When the strains were allowed to reach high GFP steady-state levels in 1 μM IP and were then reduced to sub-threshold concentrations of IP (0.012, 0.025, and 0.05 μM IP), the strains exhibited no memory and showed a monostable response (for clarity, reverse curves are not shown as they overlay the forward curves). (F) Steady-state dose–response curves for tTF strains. While the steady-state response for the cRtTF strain exhibits slightly greater ultrasensitivity (nH∼4), the dual-feedback strains sRtTF and tRtTF strikingly function as almost as pure binary switches (nH∼20). When the strains were allowed to reached high GFP steady-state levels in 1 μM IP and were then reduced to sub-threshold concentrations of IP (0.012, 0.025, and 0.05 μM IP), all three strains exhibited memory (reverse curves shown as dotted lines). (G) Deactivation kinetics for cRcTF strain. After reaching high GFP steady-state levels in 1 μM IP, strain cRcTF was thoroughly washed and resuspended in medium with different sub-threshold IP concentrations (0.012, 0.025, and 0.05 μM). The temporal deactivation curves indicate no temporal memory in this strain when compared with the 0 μM IP concentration curve (dotted gray line). (H) Deactivation kinetics for tRtTF strain. After reaching high GFP steady-state levels in 1 μM IP, strain tRtTF was thoroughly washed and resuspended in medium with different sub-threshold IP concentrations (0.012, 0.025, and 0.05 μM). For all sub-threshold concentrations, the strain shows temporal memory when compared with the 0 μM IP concentration curve (dotted gray line); however, the strain is only bistable at 0.05 μM IP. Source data is available for this figure at www.nature.com/msb.

Mentions: Increasing the receptor feedback strength (TR-SSRE>SSRE>CYC1) (Chen and Weiss, 2005) did not change the steady-state setpoint of GFP when SKN7 was only driven by the constitutive promoter (Figure 2A). This is due to the fact that receptor expression from all tested promoters was sufficient to activate the endogenous levels of SKN7; hence, these systems are all transcription factor limited (see Figure 1C). However, when we integrated an additional copy of the SKN7 gene, driven by the strong TR-SSRE promoter, the system became receptor limited. In contrast to the transcription factor-limited regime, increasing the receptor feedback strength enabled modulation of the steady-state GFP level (Figure 2B).


Synthetic conversion of a graded receptor signal into a tunable, reversible switch.

Palani S, Sarkar CA - Mol. Syst. Biol. (2011)

Feedback modulation of kinetic and steady-state system responses. (A) Activation kinetics for cTF strains. Strains cRcTF, sRcTF, and tRcTF were induced with 1 μM IP and aliquots were taken from the cultures at different time points to determine GFP expression kinetics and steady-state levels. As these strains are all transcription factor limited, the increased receptor feedback does not enhance steady-state GFP expression. (B) Activation kinetics for tTF strains. Similarly, GFP expression kinetics and steady-state levels were quantified for strains cRtTF, sRtTF, and tRtTF after induction with 1 μM IP. The strong transcription factor feedback loop makes these strains receptor limited, so receptor feedback now has a significant effect on steady-state GFP expression. (C) Temporal activation profiles of all six strains normalized to their steady-state setpoint. All six strains reach 50% of the steady-state level at different times (Act50), with stronger feedback loops slowing the kinetics. (D) Temporal deactivation profiles of the strains normalized to the steady-state setpoint. After reaching steady-state GFP expression in 1 μM IP, the cultures were thoroughly washed and resuspended in media with no IP. All strains exhibit reversibility in the absence of IP, but the deactivation kinetics are markedly different. The time at which 50% of the initial GFP expression level is reached (Deact50) is fastest for the basic and receptor feedback strains and slowest for the double feedback strains. (E) Steady-state dose–response curves for cTF strains. For cRcTF, sRcTF, and tRcTF strains, the steady-state GFP responses to IP (0.01–10 μM) are indistinguishable and weakly ultrasensitive (nH∼2). When the strains were allowed to reach high GFP steady-state levels in 1 μM IP and were then reduced to sub-threshold concentrations of IP (0.012, 0.025, and 0.05 μM IP), the strains exhibited no memory and showed a monostable response (for clarity, reverse curves are not shown as they overlay the forward curves). (F) Steady-state dose–response curves for tTF strains. While the steady-state response for the cRtTF strain exhibits slightly greater ultrasensitivity (nH∼4), the dual-feedback strains sRtTF and tRtTF strikingly function as almost as pure binary switches (nH∼20). When the strains were allowed to reached high GFP steady-state levels in 1 μM IP and were then reduced to sub-threshold concentrations of IP (0.012, 0.025, and 0.05 μM IP), all three strains exhibited memory (reverse curves shown as dotted lines). (G) Deactivation kinetics for cRcTF strain. After reaching high GFP steady-state levels in 1 μM IP, strain cRcTF was thoroughly washed and resuspended in medium with different sub-threshold IP concentrations (0.012, 0.025, and 0.05 μM). The temporal deactivation curves indicate no temporal memory in this strain when compared with the 0 μM IP concentration curve (dotted gray line). (H) Deactivation kinetics for tRtTF strain. After reaching high GFP steady-state levels in 1 μM IP, strain tRtTF was thoroughly washed and resuspended in medium with different sub-threshold IP concentrations (0.012, 0.025, and 0.05 μM). For all sub-threshold concentrations, the strain shows temporal memory when compared with the 0 μM IP concentration curve (dotted gray line); however, the strain is only bistable at 0.05 μM IP. Source data is available for this figure at www.nature.com/msb.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094063&req=5

f2: Feedback modulation of kinetic and steady-state system responses. (A) Activation kinetics for cTF strains. Strains cRcTF, sRcTF, and tRcTF were induced with 1 μM IP and aliquots were taken from the cultures at different time points to determine GFP expression kinetics and steady-state levels. As these strains are all transcription factor limited, the increased receptor feedback does not enhance steady-state GFP expression. (B) Activation kinetics for tTF strains. Similarly, GFP expression kinetics and steady-state levels were quantified for strains cRtTF, sRtTF, and tRtTF after induction with 1 μM IP. The strong transcription factor feedback loop makes these strains receptor limited, so receptor feedback now has a significant effect on steady-state GFP expression. (C) Temporal activation profiles of all six strains normalized to their steady-state setpoint. All six strains reach 50% of the steady-state level at different times (Act50), with stronger feedback loops slowing the kinetics. (D) Temporal deactivation profiles of the strains normalized to the steady-state setpoint. After reaching steady-state GFP expression in 1 μM IP, the cultures were thoroughly washed and resuspended in media with no IP. All strains exhibit reversibility in the absence of IP, but the deactivation kinetics are markedly different. The time at which 50% of the initial GFP expression level is reached (Deact50) is fastest for the basic and receptor feedback strains and slowest for the double feedback strains. (E) Steady-state dose–response curves for cTF strains. For cRcTF, sRcTF, and tRcTF strains, the steady-state GFP responses to IP (0.01–10 μM) are indistinguishable and weakly ultrasensitive (nH∼2). When the strains were allowed to reach high GFP steady-state levels in 1 μM IP and were then reduced to sub-threshold concentrations of IP (0.012, 0.025, and 0.05 μM IP), the strains exhibited no memory and showed a monostable response (for clarity, reverse curves are not shown as they overlay the forward curves). (F) Steady-state dose–response curves for tTF strains. While the steady-state response for the cRtTF strain exhibits slightly greater ultrasensitivity (nH∼4), the dual-feedback strains sRtTF and tRtTF strikingly function as almost as pure binary switches (nH∼20). When the strains were allowed to reached high GFP steady-state levels in 1 μM IP and were then reduced to sub-threshold concentrations of IP (0.012, 0.025, and 0.05 μM IP), all three strains exhibited memory (reverse curves shown as dotted lines). (G) Deactivation kinetics for cRcTF strain. After reaching high GFP steady-state levels in 1 μM IP, strain cRcTF was thoroughly washed and resuspended in medium with different sub-threshold IP concentrations (0.012, 0.025, and 0.05 μM). The temporal deactivation curves indicate no temporal memory in this strain when compared with the 0 μM IP concentration curve (dotted gray line). (H) Deactivation kinetics for tRtTF strain. After reaching high GFP steady-state levels in 1 μM IP, strain tRtTF was thoroughly washed and resuspended in medium with different sub-threshold IP concentrations (0.012, 0.025, and 0.05 μM). For all sub-threshold concentrations, the strain shows temporal memory when compared with the 0 μM IP concentration curve (dotted gray line); however, the strain is only bistable at 0.05 μM IP. Source data is available for this figure at www.nature.com/msb.
Mentions: Increasing the receptor feedback strength (TR-SSRE>SSRE>CYC1) (Chen and Weiss, 2005) did not change the steady-state setpoint of GFP when SKN7 was only driven by the constitutive promoter (Figure 2A). This is due to the fact that receptor expression from all tested promoters was sufficient to activate the endogenous levels of SKN7; hence, these systems are all transcription factor limited (see Figure 1C). However, when we integrated an additional copy of the SKN7 gene, driven by the strong TR-SSRE promoter, the system became receptor limited. In contrast to the transcription factor-limited regime, increasing the receptor feedback strength enabled modulation of the steady-state GFP level (Figure 2B).

Bottom Line: However, synthetic gene network 'switches' have been limited in their applicability and tunability due to their reliance on specific components to function.Here, we present a strategy for reversible switch design that instead relies only on a robust, easily constructed network topology with two positive feedback loops and we apply the method to create highly ultrasensitive (n(H)>20), bistable cellular responses to a synthetic ligand/receptor complex.Independent modulation of the two feedback strengths enables rational tuning and some decoupling of steady-state (ultrasensitivity, signal amplitude, switching threshold, and bistability) and kinetic (rates of system activation and deactivation) response properties.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104-6321, USA.

ABSTRACT
The ability to engineer an all-or-none cellular response to a given signaling ligand is important in applications ranging from biosensing to tissue engineering. However, synthetic gene network 'switches' have been limited in their applicability and tunability due to their reliance on specific components to function. Here, we present a strategy for reversible switch design that instead relies only on a robust, easily constructed network topology with two positive feedback loops and we apply the method to create highly ultrasensitive (n(H)>20), bistable cellular responses to a synthetic ligand/receptor complex. Independent modulation of the two feedback strengths enables rational tuning and some decoupling of steady-state (ultrasensitivity, signal amplitude, switching threshold, and bistability) and kinetic (rates of system activation and deactivation) response properties. Our integrated computational and synthetic biology approach elucidates design rules for building cellular switches with desired properties, which may be of utility in engineering signal-transduction pathways.

Show MeSH
Related in: MedlinePlus