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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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Control of the timinga-c The top, the middle and the bottom charts rows correspond to On state, Off state and On:Off ratio, respectively. The On:Off curves are calculated by dividing the fitted curves of the On by the Off measurements. a Delayed sensor characterization as a function of Cre-expressing plasmid amount. The curves are fitted to Hill function with n = 1 (On states) and to power law (Off states). b Modulation of Cre expression with an inducible promoter. ETR-iCre activity was ascertained from ETR-DsRed reporter with various concentration of erythromycin (Supplementary Fig. 4). X-axis shows percentage of promoter activity relative to full activity in absence of Erythromycin. The curves are fitted to linear (On states) and exponential (Off states) regressions. c Chemical delay of ER2-Cre-ER2 translocation. X-axis shows the time (hours) between the transfection and the addition of Tamoxifen. The curves are fitted to linear (On states) and exponential (Off states) regressions. d Recombinase cascade eliminates the leakage. Sensor diagram is shown (left). The bar chart compares standard architecture with a Cre-only delayed architecture to a cascade system, with the representative flow cytometry scatter plots shown on the right. Error bars show ± standard deviation from at least three biological replicas.
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Figure 3: Control of the timinga-c The top, the middle and the bottom charts rows correspond to On state, Off state and On:Off ratio, respectively. The On:Off curves are calculated by dividing the fitted curves of the On by the Off measurements. a Delayed sensor characterization as a function of Cre-expressing plasmid amount. The curves are fitted to Hill function with n = 1 (On states) and to power law (Off states). b Modulation of Cre expression with an inducible promoter. ETR-iCre activity was ascertained from ETR-DsRed reporter with various concentration of erythromycin (Supplementary Fig. 4). X-axis shows percentage of promoter activity relative to full activity in absence of Erythromycin. The curves are fitted to linear (On states) and exponential (Off states) regressions. c Chemical delay of ER2-Cre-ER2 translocation. X-axis shows the time (hours) between the transfection and the addition of Tamoxifen. The curves are fitted to linear (On states) and exponential (Off states) regressions. d Recombinase cascade eliminates the leakage. Sensor diagram is shown (left). The bar chart compares standard architecture with a Cre-only delayed architecture to a cascade system, with the representative flow cytometry scatter plots shown on the right. Error bars show ± standard deviation from at least three biological replicas.

Mentions: We observed that higher plasmid amount accelerated the recombination (Supplementary Fig. 2c) and proceeded to investigate the effect of recombinase-encoding plasmid dosage on the leakage and the dynamic range of the sensor. The On signal (Fig. 3a, top panel) fits the Hill function with n=1 and apparent “EC50” in the order of 2 ng regardless of the quantitation method (Supplementary Fig. 3). The Off output depended on the amount of recombinase in an almost linear fashion (Off ~ [Cre]0.77) (Fig. 3a, middle panel), resulting in On:Off ratio at lower recombinase levels reaching >2000-fold (Fig. 3a, bottom panel). Following optimization, we decided to use 5 ng of the plasmid encoding EF1α–driven iCre gene in the experiments that follow, unless indicated otherwise. The reduction in the On state at low plasmid levels of recombinase-encoding construct could be explained by reduced cotransfection efficiency of the recombinase and its target. In order to modulate Cre expression without affecting cotransfection efficiency, we used an inducible promoter driven by an ET1 transactivator whose activity can be controlled by the antibiotic erythromycin31 (Supplementary Fig. 4). Similarly to plasmid titration, modulation of Cre activity showed that the Off level increased with increased recombinase expression, but the On state was constant in the promoter activity range (Fig. 3b).


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

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

Control of the timinga-c The top, the middle and the bottom charts rows correspond to On state, Off state and On:Off ratio, respectively. The On:Off curves are calculated by dividing the fitted curves of the On by the Off measurements. a Delayed sensor characterization as a function of Cre-expressing plasmid amount. The curves are fitted to Hill function with n = 1 (On states) and to power law (Off states). b Modulation of Cre expression with an inducible promoter. ETR-iCre activity was ascertained from ETR-DsRed reporter with various concentration of erythromycin (Supplementary Fig. 4). X-axis shows percentage of promoter activity relative to full activity in absence of Erythromycin. The curves are fitted to linear (On states) and exponential (Off states) regressions. c Chemical delay of ER2-Cre-ER2 translocation. X-axis shows the time (hours) between the transfection and the addition of Tamoxifen. The curves are fitted to linear (On states) and exponential (Off states) regressions. d Recombinase cascade eliminates the leakage. Sensor diagram is shown (left). The bar chart compares standard architecture with a Cre-only delayed architecture to a cascade system, with the representative flow cytometry scatter plots shown on the right. Error bars show ± standard deviation from at least three biological replicas.
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Related In: Results  -  Collection

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Figure 3: Control of the timinga-c The top, the middle and the bottom charts rows correspond to On state, Off state and On:Off ratio, respectively. The On:Off curves are calculated by dividing the fitted curves of the On by the Off measurements. a Delayed sensor characterization as a function of Cre-expressing plasmid amount. The curves are fitted to Hill function with n = 1 (On states) and to power law (Off states). b Modulation of Cre expression with an inducible promoter. ETR-iCre activity was ascertained from ETR-DsRed reporter with various concentration of erythromycin (Supplementary Fig. 4). X-axis shows percentage of promoter activity relative to full activity in absence of Erythromycin. The curves are fitted to linear (On states) and exponential (Off states) regressions. c Chemical delay of ER2-Cre-ER2 translocation. X-axis shows the time (hours) between the transfection and the addition of Tamoxifen. The curves are fitted to linear (On states) and exponential (Off states) regressions. d Recombinase cascade eliminates the leakage. Sensor diagram is shown (left). The bar chart compares standard architecture with a Cre-only delayed architecture to a cascade system, with the representative flow cytometry scatter plots shown on the right. Error bars show ± standard deviation from at least three biological replicas.
Mentions: We observed that higher plasmid amount accelerated the recombination (Supplementary Fig. 2c) and proceeded to investigate the effect of recombinase-encoding plasmid dosage on the leakage and the dynamic range of the sensor. The On signal (Fig. 3a, top panel) fits the Hill function with n=1 and apparent “EC50” in the order of 2 ng regardless of the quantitation method (Supplementary Fig. 3). The Off output depended on the amount of recombinase in an almost linear fashion (Off ~ [Cre]0.77) (Fig. 3a, middle panel), resulting in On:Off ratio at lower recombinase levels reaching >2000-fold (Fig. 3a, bottom panel). Following optimization, we decided to use 5 ng of the plasmid encoding EF1α–driven iCre gene in the experiments that follow, unless indicated otherwise. The reduction in the On state at low plasmid levels of recombinase-encoding construct could be explained by reduced cotransfection efficiency of the recombinase and its target. In order to modulate Cre expression without affecting cotransfection efficiency, we used an inducible promoter driven by an ET1 transactivator whose activity can be controlled by the antibiotic erythromycin31 (Supplementary Fig. 4). Similarly to plasmid titration, modulation of Cre activity showed that the Off level increased with increased recombinase expression, but the On state was constant in the promoter activity range (Fig. 3b).

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