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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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Circuit blueprints of the bow-tie architectureBoth circuits are based on the same fan-in module that controls a synthetic transactivator by integrating miRNA inputs. The rtTA and LacI-based constructs sense “high” miRNA input (miR-21), while targets in the 3′-UTR of the transactivator sense three “low” miRNA (miR-141, miR-142 and miR-146a). This arrangement triggers transactivator expression only in the presence of “high” miR-21 and the absence of “low” miR-141, miR-142 and miR-146a. DNA and RNA species are lumped together, with transcriptional control occurring on DNA level while splicing and RNAi taking place on the mRNA level. a / Reversible knot configuration. The transactivator induces one or more outputs reversibly via an inducible promoter. b / Irreversible knot configuration. The transactivator induces a recombinase that irreversibly modifies one or more outputs through an appropriate switch cassette. The insets show the high-level bow-tie wiring diagrams.
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Figure 1: Circuit blueprints of the bow-tie architectureBoth circuits are based on the same fan-in module that controls a synthetic transactivator by integrating miRNA inputs. The rtTA and LacI-based constructs sense “high” miRNA input (miR-21), while targets in the 3′-UTR of the transactivator sense three “low” miRNA (miR-141, miR-142 and miR-146a). This arrangement triggers transactivator expression only in the presence of “high” miR-21 and the absence of “low” miR-141, miR-142 and miR-146a. DNA and RNA species are lumped together, with transcriptional control occurring on DNA level while splicing and RNAi taking place on the mRNA level. a / Reversible knot configuration. The transactivator induces one or more outputs reversibly via an inducible promoter. b / Irreversible knot configuration. The transactivator induces a recombinase that irreversibly modifies one or more outputs through an appropriate switch cassette. The insets show the high-level bow-tie wiring diagrams.

Mentions: In the blueprint for the proof-of-concept bow-tie circuit, the fan-in component is a variant of an RNAi classifier network28, logically integrating a number of over- and un-expressed miRNA inputs according to the logic formula “Output = miR-21 and not(miR-141) and not(miR-142) and not(miR-146a)”. Rather then converging on an output gene, the inputs fan into either of the two bow-tie knots: (i) a synthetic transactivator (Fig. 1a) or (ii) a transactivator controlling an inducible site-specific recombinase and a recombinase-triggered switch (Fig. 1b). The former implements reversible downstream actuation by concurrent induction of bow-tie outputs via an inducible promoter; the latter is used for irreversible actuation of bow-tie outputs furnished with recombinase-triggered switch elements. The circuit blueprint contains a number of unspecified components such as the transactivator, the inducible promoter, the recombinase, and the recombinase-triggered switch.


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

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

Circuit blueprints of the bow-tie architectureBoth circuits are based on the same fan-in module that controls a synthetic transactivator by integrating miRNA inputs. The rtTA and LacI-based constructs sense “high” miRNA input (miR-21), while targets in the 3′-UTR of the transactivator sense three “low” miRNA (miR-141, miR-142 and miR-146a). This arrangement triggers transactivator expression only in the presence of “high” miR-21 and the absence of “low” miR-141, miR-142 and miR-146a. DNA and RNA species are lumped together, with transcriptional control occurring on DNA level while splicing and RNAi taking place on the mRNA level. a / Reversible knot configuration. The transactivator induces one or more outputs reversibly via an inducible promoter. b / Irreversible knot configuration. The transactivator induces a recombinase that irreversibly modifies one or more outputs through an appropriate switch cassette. The insets show the high-level bow-tie wiring diagrams.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4197411&req=5

Figure 1: Circuit blueprints of the bow-tie architectureBoth circuits are based on the same fan-in module that controls a synthetic transactivator by integrating miRNA inputs. The rtTA and LacI-based constructs sense “high” miRNA input (miR-21), while targets in the 3′-UTR of the transactivator sense three “low” miRNA (miR-141, miR-142 and miR-146a). This arrangement triggers transactivator expression only in the presence of “high” miR-21 and the absence of “low” miR-141, miR-142 and miR-146a. DNA and RNA species are lumped together, with transcriptional control occurring on DNA level while splicing and RNAi taking place on the mRNA level. a / Reversible knot configuration. The transactivator induces one or more outputs reversibly via an inducible promoter. b / Irreversible knot configuration. The transactivator induces a recombinase that irreversibly modifies one or more outputs through an appropriate switch cassette. The insets show the high-level bow-tie wiring diagrams.
Mentions: In the blueprint for the proof-of-concept bow-tie circuit, the fan-in component is a variant of an RNAi classifier network28, logically integrating a number of over- and un-expressed miRNA inputs according to the logic formula “Output = miR-21 and not(miR-141) and not(miR-142) and not(miR-146a)”. Rather then converging on an output gene, the inputs fan into either of the two bow-tie knots: (i) a synthetic transactivator (Fig. 1a) or (ii) a transactivator controlling an inducible site-specific recombinase and a recombinase-triggered switch (Fig. 1b). The former implements reversible downstream actuation by concurrent induction of bow-tie outputs via an inducible promoter; the latter is used for irreversible actuation of bow-tie outputs furnished with recombinase-triggered switch elements. The circuit blueprint contains a number of unspecified components such as the transactivator, the inducible promoter, the recombinase, and the recombinase-triggered switch.

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