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Digital switching in a biosensor circuit via programmable timing of gene availability.

Lapique N, Benenson Y - Nat. Chem. Biol. (2014)

Bottom Line: Here we show that site-specific recombinases can rectify undesired effects by programmable timing of gene availability in multigene circuits.The new sensors display a dynamic range of up to 1,000-fold compared to 20-fold in the standard configuration.Our study opens new venues in gene circuit design via judicious temporal control of circuits' genetic makeup.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosystems Science and Engineering, Swiss Federal Institute of Technology Zurich (ETHZ), Basel, Switzerland.

ABSTRACT
Transient delivery of gene circuits is required in many potential applications of synthetic biology, yet the pre-steady-state processes that dominate this delivery route pose major challenges for robust circuit deployment. Here we show that site-specific recombinases can rectify undesired effects by programmable timing of gene availability in multigene circuits. We exemplify the concept with a proportional sensor for endogenous microRNA (miRNA) and show a marked reduction in its ground state leakage due to desynchronization of the circuit's repressor components and their repression target. The new sensors display a dynamic range of up to 1,000-fold compared to 20-fold in the standard configuration. We applied the approach to classify cell types on the basis of miRNA expression profile and measured >200-fold output differential between positively and negatively identified cells. We also showed major improvements in specificity with cytotoxic output. Our study opens new venues in gene circuit design via judicious temporal control of circuits' genetic makeup.

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Source of leakage in the proportional sensora General layout of a proportional sensor for a negative regulator. An input elicits inhibitory effect on a double-inversion module, which in turn negatively regulates the output. b Circuit diagram of the proportional miRNA sensor, as previously reported18. Pointed arrows indicate activation while blunted arrows denote repression. Rectangles targeted by miRNA represent four identical sites for miR-21 and three identical sites for miR-FF4. c Total output signal shown as a function of time in both On (+ miR-21 mimics) and Off (+ Neg Ctrl mimics) sensor states. The signal is the sum of all DsRed intensities in DsRed-positive cells. The signal was not normalized because the expression of the transfection marker AmCyan showed very similar dynamics in all cases. Inset, time course over 72 hours. d Mean fluorescence of DsRed-expressing cells in Off (Blue) and On (Red) states, respectively, as a function of time. The curves were drawn manually to serve as guides.
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Figure 1: Source of leakage in the proportional sensora General layout of a proportional sensor for a negative regulator. An input elicits inhibitory effect on a double-inversion module, which in turn negatively regulates the output. b Circuit diagram of the proportional miRNA sensor, as previously reported18. Pointed arrows indicate activation while blunted arrows denote repression. Rectangles targeted by miRNA represent four identical sites for miR-21 and three identical sites for miR-FF4. c Total output signal shown as a function of time in both On (+ miR-21 mimics) and Off (+ Neg Ctrl mimics) sensor states. The signal is the sum of all DsRed intensities in DsRed-positive cells. The signal was not normalized because the expression of the transfection marker AmCyan showed very similar dynamics in all cases. Inset, time course over 72 hours. d Mean fluorescence of DsRed-expressing cells in Off (Blue) and On (Red) states, respectively, as a function of time. The curves were drawn manually to serve as guides.

Mentions: Many cellular pathways are controlled by external molecular inputs via natural biosensor molecules such as cell surface receptors and transcription factors1. Engineered biosensing circuits are likewise increasingly used in basic research2,3, bioproduction4 and medicine5,6. Research in synthetic biology has greatly expanded the repertoire of tools for biosensor engineering with the development of toggle switches7,8, band-pass filters9, open-loop sensors10, riboswitches11, time-delay circuits12 and oscillators13,14. Perhaps the most basic mode of sensing is proportional sensing15, where increasing levels of input signal elicit higher levels of sensor output. For inputs with natural repressor function such as small molecule inhibitors, miRNA or transcriptional repressors, proportional sensing can be implemented with a synthetic “double inversion” module capable of suppressing the output expression while being suppressed by the input16-19 (Fig. 1a). A simple way to characterize a sensor is to measure its Off and On states in the absence and presence of an input, respectively. Large dynamic range, that is, On:Off ratio, with such sensors is achieved by exogenous ligand IPTG17, but for endogenous miRNA inputs the range is more modest18.


Digital switching in a biosensor circuit via programmable timing of gene availability.

Lapique N, Benenson Y - Nat. Chem. Biol. (2014)

Source of leakage in the proportional sensora General layout of a proportional sensor for a negative regulator. An input elicits inhibitory effect on a double-inversion module, which in turn negatively regulates the output. b Circuit diagram of the proportional miRNA sensor, as previously reported18. Pointed arrows indicate activation while blunted arrows denote repression. Rectangles targeted by miRNA represent four identical sites for miR-21 and three identical sites for miR-FF4. c Total output signal shown as a function of time in both On (+ miR-21 mimics) and Off (+ Neg Ctrl mimics) sensor states. The signal is the sum of all DsRed intensities in DsRed-positive cells. The signal was not normalized because the expression of the transfection marker AmCyan showed very similar dynamics in all cases. Inset, time course over 72 hours. d Mean fluorescence of DsRed-expressing cells in Off (Blue) and On (Red) states, respectively, as a function of time. The curves were drawn manually to serve as guides.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4232471&req=5

Figure 1: Source of leakage in the proportional sensora General layout of a proportional sensor for a negative regulator. An input elicits inhibitory effect on a double-inversion module, which in turn negatively regulates the output. b Circuit diagram of the proportional miRNA sensor, as previously reported18. Pointed arrows indicate activation while blunted arrows denote repression. Rectangles targeted by miRNA represent four identical sites for miR-21 and three identical sites for miR-FF4. c Total output signal shown as a function of time in both On (+ miR-21 mimics) and Off (+ Neg Ctrl mimics) sensor states. The signal is the sum of all DsRed intensities in DsRed-positive cells. The signal was not normalized because the expression of the transfection marker AmCyan showed very similar dynamics in all cases. Inset, time course over 72 hours. d Mean fluorescence of DsRed-expressing cells in Off (Blue) and On (Red) states, respectively, as a function of time. The curves were drawn manually to serve as guides.
Mentions: Many cellular pathways are controlled by external molecular inputs via natural biosensor molecules such as cell surface receptors and transcription factors1. Engineered biosensing circuits are likewise increasingly used in basic research2,3, bioproduction4 and medicine5,6. Research in synthetic biology has greatly expanded the repertoire of tools for biosensor engineering with the development of toggle switches7,8, band-pass filters9, open-loop sensors10, riboswitches11, time-delay circuits12 and oscillators13,14. Perhaps the most basic mode of sensing is proportional sensing15, where increasing levels of input signal elicit higher levels of sensor output. For inputs with natural repressor function such as small molecule inhibitors, miRNA or transcriptional repressors, proportional sensing can be implemented with a synthetic “double inversion” module capable of suppressing the output expression while being suppressed by the input16-19 (Fig. 1a). A simple way to characterize a sensor is to measure its Off and On states in the absence and presence of an input, respectively. Large dynamic range, that is, On:Off ratio, with such sensors is achieved by exogenous ligand IPTG17, but for endogenous miRNA inputs the range is more modest18.

Bottom Line: Here we show that site-specific recombinases can rectify undesired effects by programmable timing of gene availability in multigene circuits.The new sensors display a dynamic range of up to 1,000-fold compared to 20-fold in the standard configuration.Our study opens new venues in gene circuit design via judicious temporal control of circuits' genetic makeup.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosystems Science and Engineering, Swiss Federal Institute of Technology Zurich (ETHZ), Basel, Switzerland.

ABSTRACT
Transient delivery of gene circuits is required in many potential applications of synthetic biology, yet the pre-steady-state processes that dominate this delivery route pose major challenges for robust circuit deployment. Here we show that site-specific recombinases can rectify undesired effects by programmable timing of gene availability in multigene circuits. We exemplify the concept with a proportional sensor for endogenous microRNA (miRNA) and show a marked reduction in its ground state leakage due to desynchronization of the circuit's repressor components and their repression target. The new sensors display a dynamic range of up to 1,000-fold compared to 20-fold in the standard configuration. We applied the approach to classify cell types on the basis of miRNA expression profile and measured >200-fold output differential between positively and negatively identified cells. We also showed major improvements in specificity with cytotoxic output. Our study opens new venues in gene circuit design via judicious temporal control of circuits' genetic makeup.

Show MeSH
Related in: MedlinePlus