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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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Second-iteration bow-tie circuit instantiation and preliminary characterizationa / Detailed schematics of second-iteration circuits: irreversible V2-FlpO (top) and reversible V2-Rev (bottom). DNA and RNA species are lumped together, with transcriptional regulation and recombination taking place at the DNA level and splicing/RNAi at the RNA level. Different genetic building blocks are indicated. The insets show the high-level diagrams of the corresponding bow-tie architectures. b / Dose response of the On and Off readouts and the On:Off ratio to the amount of sensor-encoding plasmids in V2-FlpO. The amounts of miR-21 sensor genes are changed simultaneously, while all the other components are kept constant. Left and right charts show the changes in Cerulean and Citrine readouts, respectively. Both charts show mean±SD of three biological replicates with manually drawn curves serving as visual guides. Error propagation rules were applied to calculate the SD of the ratio. Plasmid amounts are provided in Supplementary Table 5.
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Figure 4: Second-iteration bow-tie circuit instantiation and preliminary characterizationa / Detailed schematics of second-iteration circuits: irreversible V2-FlpO (top) and reversible V2-Rev (bottom). DNA and RNA species are lumped together, with transcriptional regulation and recombination taking place at the DNA level and splicing/RNAi at the RNA level. Different genetic building blocks are indicated. The insets show the high-level diagrams of the corresponding bow-tie architectures. b / Dose response of the On and Off readouts and the On:Off ratio to the amount of sensor-encoding plasmids in V2-FlpO. The amounts of miR-21 sensor genes are changed simultaneously, while all the other components are kept constant. Left and right charts show the changes in Cerulean and Citrine readouts, respectively. Both charts show mean±SD of three biological replicates with manually drawn curves serving as visual guides. Error propagation rules were applied to calculate the SD of the ratio. Plasmid amounts are provided in Supplementary Table 5.

Mentions: Next, we placed the miR-21 sensor upstream of the knot in order to characterize circuit readouts under varying input conditions. We varied the dosage of sensor components and used miR-21 and negative control miRNA mimics to generate On and Off sensor states, respectively (Fig. 3c, Supplementary Table 4b). We found that the On:Off ratios, observed at the Cerulean/PIT2 level, deteriorated at the Citrine level due to amplification of the PIT2 leakage in the Off state. The best ratios were obtained with 120 ng of each sensor gene resulting in overall dosage ratio of 10:10:1:1:10 in the cascade. The low On:Off ratios highlighted the importance of leakage suppression during signal transduction from the fan-in to the fan-out modules in the irreversible knot. In order to reduce this leakage, an approach was developed (Lapique et al., manuscript in revision) whereby the miRNA sensor output itself is incorporated in a Flex switch in an inverted orientation. The orientation is restored by a constitutively-expressed recombinase, resulting in a delay relative to the expression of sensor genes and leading to accumulation of repressor species resulting in much lower Off state. We utilized this method, flanking the inverted Cerulean-2A-PIT2 by loxP and lox2272 sites as in Cre-Flex switch and providing EF1a-driven Cre in trans. FlpO replaced Cre in the irreversible knot resulting in PIT2/pPIRtight-FlpO/CMV-CIitrineFlpO-Flex knot configuration and a circuit labeled V2-FlpO. We also constructed its reversible analog, V2-Rev, where PIT2 directly controls output proteins via pPIRtight promoter (Fig. 4a). MiR-21 sensor characterization with V2-FlpO showed large improvement in both Cerulean and Citrine On:Off ratios (Fig. 4b, Supplementary Table 5), as expected.


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

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

Second-iteration bow-tie circuit instantiation and preliminary characterizationa / Detailed schematics of second-iteration circuits: irreversible V2-FlpO (top) and reversible V2-Rev (bottom). DNA and RNA species are lumped together, with transcriptional regulation and recombination taking place at the DNA level and splicing/RNAi at the RNA level. Different genetic building blocks are indicated. The insets show the high-level diagrams of the corresponding bow-tie architectures. b / Dose response of the On and Off readouts and the On:Off ratio to the amount of sensor-encoding plasmids in V2-FlpO. The amounts of miR-21 sensor genes are changed simultaneously, while all the other components are kept constant. Left and right charts show the changes in Cerulean and Citrine readouts, respectively. Both charts show mean±SD of three biological replicates with manually drawn curves serving as visual guides. Error propagation rules were applied to calculate the SD of the ratio. Plasmid amounts are provided in Supplementary Table 5.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4197411&req=5

Figure 4: Second-iteration bow-tie circuit instantiation and preliminary characterizationa / Detailed schematics of second-iteration circuits: irreversible V2-FlpO (top) and reversible V2-Rev (bottom). DNA and RNA species are lumped together, with transcriptional regulation and recombination taking place at the DNA level and splicing/RNAi at the RNA level. Different genetic building blocks are indicated. The insets show the high-level diagrams of the corresponding bow-tie architectures. b / Dose response of the On and Off readouts and the On:Off ratio to the amount of sensor-encoding plasmids in V2-FlpO. The amounts of miR-21 sensor genes are changed simultaneously, while all the other components are kept constant. Left and right charts show the changes in Cerulean and Citrine readouts, respectively. Both charts show mean±SD of three biological replicates with manually drawn curves serving as visual guides. Error propagation rules were applied to calculate the SD of the ratio. Plasmid amounts are provided in Supplementary Table 5.
Mentions: Next, we placed the miR-21 sensor upstream of the knot in order to characterize circuit readouts under varying input conditions. We varied the dosage of sensor components and used miR-21 and negative control miRNA mimics to generate On and Off sensor states, respectively (Fig. 3c, Supplementary Table 4b). We found that the On:Off ratios, observed at the Cerulean/PIT2 level, deteriorated at the Citrine level due to amplification of the PIT2 leakage in the Off state. The best ratios were obtained with 120 ng of each sensor gene resulting in overall dosage ratio of 10:10:1:1:10 in the cascade. The low On:Off ratios highlighted the importance of leakage suppression during signal transduction from the fan-in to the fan-out modules in the irreversible knot. In order to reduce this leakage, an approach was developed (Lapique et al., manuscript in revision) whereby the miRNA sensor output itself is incorporated in a Flex switch in an inverted orientation. The orientation is restored by a constitutively-expressed recombinase, resulting in a delay relative to the expression of sensor genes and leading to accumulation of repressor species resulting in much lower Off state. We utilized this method, flanking the inverted Cerulean-2A-PIT2 by loxP and lox2272 sites as in Cre-Flex switch and providing EF1a-driven Cre in trans. FlpO replaced Cre in the irreversible knot resulting in PIT2/pPIRtight-FlpO/CMV-CIitrineFlpO-Flex knot configuration and a circuit labeled V2-FlpO. We also constructed its reversible analog, V2-Rev, where PIT2 directly controls output proteins via pPIRtight promoter (Fig. 4a). MiR-21 sensor characterization with V2-FlpO showed large improvement in both Cerulean and Citrine On:Off ratios (Fig. 4b, Supplementary Table 5), as expected.

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