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Highly modular bow-tie gene circuits with programmable dynamic behaviour.

Prochazka L, Angelici B, Haefliger B, Benenson Y - Nat Commun (2014)

Bottom Line: Synthetic gene circuits often require extensive mutual optimization of their components for successful operation, while modular and programmable design platforms are rare.We characterize the circuits in HEK293 cells, confirming their modularity and scalability, and validate them using endogenous microRNA inputs in additional cell lines.This platform can be used for biotechnological and biomedical applications in vitro, in vivo and potentially in human therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosystems Science and Engineering (D-BSSE), Swiss Federal Institute of Technology (ETH) Zürich, Mattenstrasse 26, Basel 4058, Switzerland.

ABSTRACT
Synthetic gene circuits often require extensive mutual optimization of their components for successful operation, while modular and programmable design platforms are rare. A possible solution lies in the 'bow-tie' architecture, which stipulates a focal component-a 'knot'-uncoupling circuits' inputs and outputs, simplifying component swapping, and introducing additional layer of control. Here we construct, in cultured human cells, synthetic bow-tie circuits that transduce microRNA inputs into protein outputs with independently programmable logical and dynamic behaviour. The latter is adjusted via two different knot configurations: a transcriptional activator causing the outputs to track input changes reversibly, and a recombinase-based cascade, converting transient inputs into permanent actuation. We characterize the circuits in HEK293 cells, confirming their modularity and scalability, and validate them using endogenous microRNA inputs in additional cell lines. This platform can be used for biotechnological and biomedical applications in vitro, in vivo and potentially in human therapy.

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Characterization of second-iteration circuits in microRNA profiling taska / Detailed circuits and their code names. Cerulean and Citrine readouts are highlighted. Shaded components with callouts in V2-FlpO diagram are removed one at a time in control experiments performed with this and other circuits. b / Simplified diagrams showing complete circuits and various controls in the context of the bow-tie architecture corresponding to irreversible (top four) and reversible (bottom four) variants. c / Experimental data. Each of the four circuits were transfected together with different miRNA input combinations as indicated in the tables on the left. The bar charts show normalized Cerulean and Citrine intensities as mean±SD from three independent biological replicates. Representative microscopy snapshots show the Citrine expression (Yellow pseudocolor) and the transfection marker mCherry expression (Red pseudocolor). Cerulean snapshots are not shown due to very low Cerulean signal because of low plasmid dosage. Two-sided unpaired t-tests were performed for observed differences in Citrine in the Off-configuration samples (−/−, +/+, −/+) compared to the On configuration (+/−). P-values are indicated as follows: ** = p<0.000001, * = p<0.0001. Transfection setup is given in Supplementary Table 6 and quantitative values in Supplementary Table 7. Representative flow cytometry plots and raw data are in Supplementary Fig. 5.
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Figure 5: Characterization of second-iteration circuits in microRNA profiling taska / Detailed circuits and their code names. Cerulean and Citrine readouts are highlighted. Shaded components with callouts in V2-FlpO diagram are removed one at a time in control experiments performed with this and other circuits. b / Simplified diagrams showing complete circuits and various controls in the context of the bow-tie architecture corresponding to irreversible (top four) and reversible (bottom four) variants. c / Experimental data. Each of the four circuits were transfected together with different miRNA input combinations as indicated in the tables on the left. The bar charts show normalized Cerulean and Citrine intensities as mean±SD from three independent biological replicates. Representative microscopy snapshots show the Citrine expression (Yellow pseudocolor) and the transfection marker mCherry expression (Red pseudocolor). Cerulean snapshots are not shown due to very low Cerulean signal because of low plasmid dosage. Two-sided unpaired t-tests were performed for observed differences in Citrine in the Off-configuration samples (−/−, +/+, −/+) compared to the On configuration (+/−). P-values are indicated as follows: ** = p<0.000001, * = p<0.0001. Transfection setup is given in Supplementary Table 6 and quantitative values in Supplementary Table 7. Representative flow cytometry plots and raw data are in Supplementary Fig. 5.

Mentions: In order to perform comprehensive circuits’ characterization, we first tested them in a miRNA profiling task, measuring their output at a fixed time-point with different fan-in logical input combinations using miRNA mimics in HEK293 cells, whereby the input is either fully active (+) or completely inactive (−). We used miR-21 as the “high” input and a mixture of miR-141 and miR-146a as a composite “low” input, resulting in four possible combinations. The On state should only occur in the presence of miR-21 and absence of miR-141 and miR-146a (combination +/−), while the Off state is expected to occur in three other cases. Additionally, we included two negative controls for each circuit: one without Cerulean-2A-PIT2 (ΔPIT2), to measure leakage from pPIRtight, another without PIR-driven construct (ΔPIR) to confirm the central role of this promoter; and a positive control without miR-21 sensor (ΔS21) to determine maximal induction of the output. Furthermore, to quantify the effect of flexed PIT2 cassette, we assembled circuits containing pre-recombined, forward-facing Cerulean-2A-PIT2 genes denoted as V2-FlpO-Fwd and V2-Rev-Fwd and their corresponding controls (Fig. 5a,b).


Highly modular bow-tie gene circuits with programmable dynamic behaviour.

Prochazka L, Angelici B, Haefliger B, Benenson Y - Nat Commun (2014)

Characterization of second-iteration circuits in microRNA profiling taska / Detailed circuits and their code names. Cerulean and Citrine readouts are highlighted. Shaded components with callouts in V2-FlpO diagram are removed one at a time in control experiments performed with this and other circuits. b / Simplified diagrams showing complete circuits and various controls in the context of the bow-tie architecture corresponding to irreversible (top four) and reversible (bottom four) variants. c / Experimental data. Each of the four circuits were transfected together with different miRNA input combinations as indicated in the tables on the left. The bar charts show normalized Cerulean and Citrine intensities as mean±SD from three independent biological replicates. Representative microscopy snapshots show the Citrine expression (Yellow pseudocolor) and the transfection marker mCherry expression (Red pseudocolor). Cerulean snapshots are not shown due to very low Cerulean signal because of low plasmid dosage. Two-sided unpaired t-tests were performed for observed differences in Citrine in the Off-configuration samples (−/−, +/+, −/+) compared to the On configuration (+/−). P-values are indicated as follows: ** = p<0.000001, * = p<0.0001. Transfection setup is given in Supplementary Table 6 and quantitative values in Supplementary Table 7. Representative flow cytometry plots and raw data are in Supplementary Fig. 5.
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Figure 5: Characterization of second-iteration circuits in microRNA profiling taska / Detailed circuits and their code names. Cerulean and Citrine readouts are highlighted. Shaded components with callouts in V2-FlpO diagram are removed one at a time in control experiments performed with this and other circuits. b / Simplified diagrams showing complete circuits and various controls in the context of the bow-tie architecture corresponding to irreversible (top four) and reversible (bottom four) variants. c / Experimental data. Each of the four circuits were transfected together with different miRNA input combinations as indicated in the tables on the left. The bar charts show normalized Cerulean and Citrine intensities as mean±SD from three independent biological replicates. Representative microscopy snapshots show the Citrine expression (Yellow pseudocolor) and the transfection marker mCherry expression (Red pseudocolor). Cerulean snapshots are not shown due to very low Cerulean signal because of low plasmid dosage. Two-sided unpaired t-tests were performed for observed differences in Citrine in the Off-configuration samples (−/−, +/+, −/+) compared to the On configuration (+/−). P-values are indicated as follows: ** = p<0.000001, * = p<0.0001. Transfection setup is given in Supplementary Table 6 and quantitative values in Supplementary Table 7. Representative flow cytometry plots and raw data are in Supplementary Fig. 5.
Mentions: In order to perform comprehensive circuits’ characterization, we first tested them in a miRNA profiling task, measuring their output at a fixed time-point with different fan-in logical input combinations using miRNA mimics in HEK293 cells, whereby the input is either fully active (+) or completely inactive (−). We used miR-21 as the “high” input and a mixture of miR-141 and miR-146a as a composite “low” input, resulting in four possible combinations. The On state should only occur in the presence of miR-21 and absence of miR-141 and miR-146a (combination +/−), while the Off state is expected to occur in three other cases. Additionally, we included two negative controls for each circuit: one without Cerulean-2A-PIT2 (ΔPIT2), to measure leakage from pPIRtight, another without PIR-driven construct (ΔPIR) to confirm the central role of this promoter; and a positive control without miR-21 sensor (ΔS21) to determine maximal induction of the output. Furthermore, to quantify the effect of flexed PIT2 cassette, we assembled circuits containing pre-recombined, forward-facing Cerulean-2A-PIT2 genes denoted as V2-FlpO-Fwd and V2-Rev-Fwd and their corresponding controls (Fig. 5a,b).

Bottom Line: Synthetic gene circuits often require extensive mutual optimization of their components for successful operation, while modular and programmable design platforms are rare.We characterize the circuits in HEK293 cells, confirming their modularity and scalability, and validate them using endogenous microRNA inputs in additional cell lines.This platform can be used for biotechnological and biomedical applications in vitro, in vivo and potentially in human therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosystems Science and Engineering (D-BSSE), Swiss Federal Institute of Technology (ETH) Zürich, Mattenstrasse 26, Basel 4058, Switzerland.

ABSTRACT
Synthetic gene circuits often require extensive mutual optimization of their components for successful operation, while modular and programmable design platforms are rare. A possible solution lies in the 'bow-tie' architecture, which stipulates a focal component-a 'knot'-uncoupling circuits' inputs and outputs, simplifying component swapping, and introducing additional layer of control. Here we construct, in cultured human cells, synthetic bow-tie circuits that transduce microRNA inputs into protein outputs with independently programmable logical and dynamic behaviour. The latter is adjusted via two different knot configurations: a transcriptional activator causing the outputs to track input changes reversibly, and a recombinase-based cascade, converting transient inputs into permanent actuation. We characterize the circuits in HEK293 cells, confirming their modularity and scalability, and validate them using endogenous microRNA inputs in additional cell lines. This platform can be used for biotechnological and biomedical applications in vitro, in vivo and potentially in human therapy.

Show MeSH
Related in: MedlinePlus