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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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Fan-in/Fan-out circuitsLeft, schematics of the circuits V2-FlpO-fan-out and V2-Rev-fan-out. Right, corresponding quantitative data next to the single-output circuit V2-FlpO and V2-Rev used for comparison. Presence or absence of miRNA mimics is indicated on the bottom of the bar charts. Cerulean, Citrine and DsRed readouts are shown as mean±SD from three independent biological replicates; microscopy snapshots show Citrine (Yellow pseudocolor), DsRed (Red pseudocolor) and iRFP expression (magenta pseudocolor) and are given for On states (+/−) only. Two-sided unpaired t-tests were performed for observed differential output expression in On and Off states. Samples that have p-values < 0.001 in both Citrine and DsRed readouts are indicated. Transfection setup is given in Supplementary Table 10, quantitative values in Supplementary Table 11 and scatter plots as well as raw data are in Supplementary Fig. 8.
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Figure 6: Fan-in/Fan-out circuitsLeft, schematics of the circuits V2-FlpO-fan-out and V2-Rev-fan-out. Right, corresponding quantitative data next to the single-output circuit V2-FlpO and V2-Rev used for comparison. Presence or absence of miRNA mimics is indicated on the bottom of the bar charts. Cerulean, Citrine and DsRed readouts are shown as mean±SD from three independent biological replicates; microscopy snapshots show Citrine (Yellow pseudocolor), DsRed (Red pseudocolor) and iRFP expression (magenta pseudocolor) and are given for On states (+/−) only. Two-sided unpaired t-tests were performed for observed differential output expression in On and Off states. Samples that have p-values < 0.001 in both Citrine and DsRed readouts are indicated. Transfection setup is given in Supplementary Table 10, quantitative values in Supplementary Table 11 and scatter plots as well as raw data are in Supplementary Fig. 8.

Mentions: Using the leakage-reducing strategy with expression-delayed, flexed Cerulean-2A-PIT2 construct in conjunction with the extensive optimization of the remaining components resulted in robust system performance. We proceeded to establish the complete bow-tie architecture with the knot controlling multiple outputs. We added output constructs expressing red fluorescent protein (pPIRtight-DsRed and CMV-DsRedFlpO-Flex), generating V2-Rev-fan-out and V2-FlpO-fan-out circuits, each controlling two independent outputs. The levels of Citrine and DsRed outputs were measured 48 hours after transfection in a miRNA profiling task in a manner similar to single-output circuits. Since we used an iRFP (infrared fluorescent protein) as a transfection control in these experiments, we also re-measured V2-Rev and V2-FlpO circuits for comparison. Both V2-Rev-fan-out and V2-FlpO-fan-out circuits could simultaneously control both outputs in a manner consistent with miRNA input combinations (Fig. 6, Supplementary Fig. 8, Supplementary Tables 10, 11).


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

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

Fan-in/Fan-out circuitsLeft, schematics of the circuits V2-FlpO-fan-out and V2-Rev-fan-out. Right, corresponding quantitative data next to the single-output circuit V2-FlpO and V2-Rev used for comparison. Presence or absence of miRNA mimics is indicated on the bottom of the bar charts. Cerulean, Citrine and DsRed readouts are shown as mean±SD from three independent biological replicates; microscopy snapshots show Citrine (Yellow pseudocolor), DsRed (Red pseudocolor) and iRFP expression (magenta pseudocolor) and are given for On states (+/−) only. Two-sided unpaired t-tests were performed for observed differential output expression in On and Off states. Samples that have p-values < 0.001 in both Citrine and DsRed readouts are indicated. Transfection setup is given in Supplementary Table 10, quantitative values in Supplementary Table 11 and scatter plots as well as raw data are in Supplementary Fig. 8.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4197411&req=5

Figure 6: Fan-in/Fan-out circuitsLeft, schematics of the circuits V2-FlpO-fan-out and V2-Rev-fan-out. Right, corresponding quantitative data next to the single-output circuit V2-FlpO and V2-Rev used for comparison. Presence or absence of miRNA mimics is indicated on the bottom of the bar charts. Cerulean, Citrine and DsRed readouts are shown as mean±SD from three independent biological replicates; microscopy snapshots show Citrine (Yellow pseudocolor), DsRed (Red pseudocolor) and iRFP expression (magenta pseudocolor) and are given for On states (+/−) only. Two-sided unpaired t-tests were performed for observed differential output expression in On and Off states. Samples that have p-values < 0.001 in both Citrine and DsRed readouts are indicated. Transfection setup is given in Supplementary Table 10, quantitative values in Supplementary Table 11 and scatter plots as well as raw data are in Supplementary Fig. 8.
Mentions: Using the leakage-reducing strategy with expression-delayed, flexed Cerulean-2A-PIT2 construct in conjunction with the extensive optimization of the remaining components resulted in robust system performance. We proceeded to establish the complete bow-tie architecture with the knot controlling multiple outputs. We added output constructs expressing red fluorescent protein (pPIRtight-DsRed and CMV-DsRedFlpO-Flex), generating V2-Rev-fan-out and V2-FlpO-fan-out circuits, each controlling two independent outputs. The levels of Citrine and DsRed outputs were measured 48 hours after transfection in a miRNA profiling task in a manner similar to single-output circuits. Since we used an iRFP (infrared fluorescent protein) as a transfection control in these experiments, we also re-measured V2-Rev and V2-FlpO circuits for comparison. Both V2-Rev-fan-out and V2-FlpO-fan-out circuits could simultaneously control both outputs in a manner consistent with miRNA input combinations (Fig. 6, Supplementary Fig. 8, Supplementary Tables 10, 11).

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