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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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The effect of delayed output on sensor performancea The principle behind the FLEx inversion system30. b-e Time course measurements of standard and delayed sensor architectures. b Time course of total output protein expression from the uncontrolled forward-facing output gene (Standard, Top, blue line) and delayed output expression from uncontrolled backward-facing output gene (Delayed, Top, red line), showing about a six-hour delay. Comparison of the On signal development in the standard (Standard, On, green line) and the delayed (Delayed, On, orange line) sensor displayed an 8-hour delay. Only the first 14 hours are shown. c Time course of total DsRed signal corresponding to the uncontrolled output gene (Top), miR-21 sensor in the presence of miR-21 mimic (On) and sensor in the presence of negative control mimic (Off), corresponding to both standard and delayed configurations as indicated in the chart, over 72 hours. The fold-ratios between the On and the Off signals are shown. The blue curve corresponds to the Off state of the standard sensor in the absence of any miRNA mimic (see main text). d Time-dependent histograms of DsRed output in the standard sensor configuration. Different setups are indicated on top. The label “Upper output bound” corresponds to DsRed expression from uncontrolled output gene (same as “Top” in b and c). e Time course measurements in the delayed configuration. The Off and On time series are as in d.
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Figure 2: The effect of delayed output on sensor performancea The principle behind the FLEx inversion system30. b-e Time course measurements of standard and delayed sensor architectures. b Time course of total output protein expression from the uncontrolled forward-facing output gene (Standard, Top, blue line) and delayed output expression from uncontrolled backward-facing output gene (Delayed, Top, red line), showing about a six-hour delay. Comparison of the On signal development in the standard (Standard, On, green line) and the delayed (Delayed, On, orange line) sensor displayed an 8-hour delay. Only the first 14 hours are shown. c Time course of total DsRed signal corresponding to the uncontrolled output gene (Top), miR-21 sensor in the presence of miR-21 mimic (On) and sensor in the presence of negative control mimic (Off), corresponding to both standard and delayed configurations as indicated in the chart, over 72 hours. The fold-ratios between the On and the Off signals are shown. The blue curve corresponds to the Off state of the standard sensor in the absence of any miRNA mimic (see main text). d Time-dependent histograms of DsRed output in the standard sensor configuration. Different setups are indicated on top. The label “Upper output bound” corresponds to DsRed expression from uncontrolled output gene (same as “Top” in b and c). e Time course measurements in the delayed configuration. The Off and On time series are as in d.

Mentions: A number of approaches could be envisioned to delay output production12,29. While control over gene expression timing using transcriptional regulation is one option, the most drastic and at the same time, “clean” intervention would be to withhold the gene itself. This requires programmable, time-dependent modification in the circuit-encoding DNA that is best achieved through DNA-manipulating enzymes such as recombinases. Therefore we opted for a recombinase-driven inversion of the output-coding region relative to its promoter. A Cre/Lox recombination system was engineered to delay output production, utilizing the FLEx switch that exploits the dual inversion and excision activity of the recombinase30. Using two pairs of incompatible Lox recombination sites, the coding region of the output sequence is stably and irreversibly inverted after Cre-mediated recombination (Fig. 2a). In a “delayed” sensor, the standard output construct was replaced with a backward-facing DsRed coding region; a constitutive Cre expression cassette was included with other circuit components. The delay amounted to 6-8 hours (Fig. 2b). As a result, we observed dramatic effect on the sensor Off state (Figs. 2c-e), in which virtually all the fluorescent leakage was eliminated, while the On state was only moderately reduced. This reduction in the On state could be largely explained by incomplete inversion of the backward-facing output (Supplementary Fig. 2a,b). There was an overall improvement of about 30-fold in the dynamic range of the sensor as shown in Fig. 2c. We note parenthetically that cotransfection of negative control mimic, which is the appropriate Off measurement while using miR-21 mimic, increased the Off state of the standard sensor and to a less extent in the delayed sensor. Yet even when the negative control mimic was withheld (blue curve in Fig. 2c), there was about an order of magnitude improvement in the Off state.


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

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

The effect of delayed output on sensor performancea The principle behind the FLEx inversion system30. b-e Time course measurements of standard and delayed sensor architectures. b Time course of total output protein expression from the uncontrolled forward-facing output gene (Standard, Top, blue line) and delayed output expression from uncontrolled backward-facing output gene (Delayed, Top, red line), showing about a six-hour delay. Comparison of the On signal development in the standard (Standard, On, green line) and the delayed (Delayed, On, orange line) sensor displayed an 8-hour delay. Only the first 14 hours are shown. c Time course of total DsRed signal corresponding to the uncontrolled output gene (Top), miR-21 sensor in the presence of miR-21 mimic (On) and sensor in the presence of negative control mimic (Off), corresponding to both standard and delayed configurations as indicated in the chart, over 72 hours. The fold-ratios between the On and the Off signals are shown. The blue curve corresponds to the Off state of the standard sensor in the absence of any miRNA mimic (see main text). d Time-dependent histograms of DsRed output in the standard sensor configuration. Different setups are indicated on top. The label “Upper output bound” corresponds to DsRed expression from uncontrolled output gene (same as “Top” in b and c). e Time course measurements in the delayed configuration. The Off and On time series are as in d.
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Figure 2: The effect of delayed output on sensor performancea The principle behind the FLEx inversion system30. b-e Time course measurements of standard and delayed sensor architectures. b Time course of total output protein expression from the uncontrolled forward-facing output gene (Standard, Top, blue line) and delayed output expression from uncontrolled backward-facing output gene (Delayed, Top, red line), showing about a six-hour delay. Comparison of the On signal development in the standard (Standard, On, green line) and the delayed (Delayed, On, orange line) sensor displayed an 8-hour delay. Only the first 14 hours are shown. c Time course of total DsRed signal corresponding to the uncontrolled output gene (Top), miR-21 sensor in the presence of miR-21 mimic (On) and sensor in the presence of negative control mimic (Off), corresponding to both standard and delayed configurations as indicated in the chart, over 72 hours. The fold-ratios between the On and the Off signals are shown. The blue curve corresponds to the Off state of the standard sensor in the absence of any miRNA mimic (see main text). d Time-dependent histograms of DsRed output in the standard sensor configuration. Different setups are indicated on top. The label “Upper output bound” corresponds to DsRed expression from uncontrolled output gene (same as “Top” in b and c). e Time course measurements in the delayed configuration. The Off and On time series are as in d.
Mentions: A number of approaches could be envisioned to delay output production12,29. While control over gene expression timing using transcriptional regulation is one option, the most drastic and at the same time, “clean” intervention would be to withhold the gene itself. This requires programmable, time-dependent modification in the circuit-encoding DNA that is best achieved through DNA-manipulating enzymes such as recombinases. Therefore we opted for a recombinase-driven inversion of the output-coding region relative to its promoter. A Cre/Lox recombination system was engineered to delay output production, utilizing the FLEx switch that exploits the dual inversion and excision activity of the recombinase30. Using two pairs of incompatible Lox recombination sites, the coding region of the output sequence is stably and irreversibly inverted after Cre-mediated recombination (Fig. 2a). In a “delayed” sensor, the standard output construct was replaced with a backward-facing DsRed coding region; a constitutive Cre expression cassette was included with other circuit components. The delay amounted to 6-8 hours (Fig. 2b). As a result, we observed dramatic effect on the sensor Off state (Figs. 2c-e), in which virtually all the fluorescent leakage was eliminated, while the On state was only moderately reduced. This reduction in the On state could be largely explained by incomplete inversion of the backward-facing output (Supplementary Fig. 2a,b). There was an overall improvement of about 30-fold in the dynamic range of the sensor as shown in Fig. 2c. We note parenthetically that cotransfection of negative control mimic, which is the appropriate Off measurement while using miR-21 mimic, increased the Off state of the standard sensor and to a less extent in the delayed sensor. Yet even when the negative control mimic was withheld (blue curve in Fig. 2c), there was about an order of magnitude improvement in the Off state.

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