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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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Performance of irreversible recombinase switchesa / Schematics of the four tested recombinase switches based on site-specific recombinase Cre, FlpO and PhiC31. The switch constructs undergo site-specific recombination in order to express constitutively-driven reporter gene through various strategies (see also Supplementary Fig.1). Triangles indicate recombinase recognition sites. b / Maximal switch response (yellow bars) and corresponding efficiency (blue bars) of each switch. The efficiency is calculated as the ratio between recombinase-triggered reporter expression level and the expression level of pre-recombined control reporter in the presence of the same recombinase amount. Each bar represents mean±SD from three biological replicates measured 48 hours after transfection by flow cytometry (see also Supplementary Fig. 1). Plasmid amounts are provided in Supplementary Table 1. c / Schematics of the experimental setup for testing regulated recombinase activity. A CMV-driven transactivator is co-transfected with a recombinase driven by an appropriate inducible promoter and a recombinase-triggered switch. d / Top: activator-promoter combinations. Bottom: bar charts comparing the basal switch response (“−”, no activator) and the maximal response observed with different transactivators (“+”) (see also Supplementary Fig. 2). Each bar represents mean±SD of three biological replicates measured 48 hours after transfection by flow cytometry. Plasmid amounts are provided in Supplementary Table 2 and quantitative readouts of key samples in Supplementary Table 3.
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Figure 2: Performance of irreversible recombinase switchesa / Schematics of the four tested recombinase switches based on site-specific recombinase Cre, FlpO and PhiC31. The switch constructs undergo site-specific recombination in order to express constitutively-driven reporter gene through various strategies (see also Supplementary Fig.1). Triangles indicate recombinase recognition sites. b / Maximal switch response (yellow bars) and corresponding efficiency (blue bars) of each switch. The efficiency is calculated as the ratio between recombinase-triggered reporter expression level and the expression level of pre-recombined control reporter in the presence of the same recombinase amount. Each bar represents mean±SD from three biological replicates measured 48 hours after transfection by flow cytometry (see also Supplementary Fig. 1). Plasmid amounts are provided in Supplementary Table 1. c / Schematics of the experimental setup for testing regulated recombinase activity. A CMV-driven transactivator is co-transfected with a recombinase driven by an appropriate inducible promoter and a recombinase-triggered switch. d / Top: activator-promoter combinations. Bottom: bar charts comparing the basal switch response (“−”, no activator) and the maximal response observed with different transactivators (“+”) (see also Supplementary Fig. 2). Each bar represents mean±SD of three biological replicates measured 48 hours after transfection by flow cytometry. Plasmid amounts are provided in Supplementary Table 2 and quantitative readouts of key samples in Supplementary Table 3.

Mentions: We first developed the irreversible knot that requires efficient recombination combined with irreversible genetic switch, and tight control of the recombinase by the transactivator to prevent uncontrolled switching and resulting output expression. We tested iCre42, FlpO43 and PhiC31o43 enzymes with several irreversible switches, including PhiC31-inversion44, Cre-excision45, Cre-Flex46 and FlpO-Flex47 (Fig. 2a, Supplementary Fig. 1a,b). We measured switches’ response to increasing dose of constitutively-expressed cognate recombinase and compared them to pre-recombined positive controls to estimate switching efficiency (Supplementary Fig. 1c, Supplementary Table 1, Supplementary Note 1). We found that all switches responded strongly to catalytic recombinase amounts. However only Cre-Flex and FlpO-Flex showed high absolute response and efficiency, and were retained for circuit development (Fig. 2b).


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

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

Performance of irreversible recombinase switchesa / Schematics of the four tested recombinase switches based on site-specific recombinase Cre, FlpO and PhiC31. The switch constructs undergo site-specific recombination in order to express constitutively-driven reporter gene through various strategies (see also Supplementary Fig.1). Triangles indicate recombinase recognition sites. b / Maximal switch response (yellow bars) and corresponding efficiency (blue bars) of each switch. The efficiency is calculated as the ratio between recombinase-triggered reporter expression level and the expression level of pre-recombined control reporter in the presence of the same recombinase amount. Each bar represents mean±SD from three biological replicates measured 48 hours after transfection by flow cytometry (see also Supplementary Fig. 1). Plasmid amounts are provided in Supplementary Table 1. c / Schematics of the experimental setup for testing regulated recombinase activity. A CMV-driven transactivator is co-transfected with a recombinase driven by an appropriate inducible promoter and a recombinase-triggered switch. d / Top: activator-promoter combinations. Bottom: bar charts comparing the basal switch response (“−”, no activator) and the maximal response observed with different transactivators (“+”) (see also Supplementary Fig. 2). Each bar represents mean±SD of three biological replicates measured 48 hours after transfection by flow cytometry. Plasmid amounts are provided in Supplementary Table 2 and quantitative readouts of key samples in Supplementary Table 3.
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Figure 2: Performance of irreversible recombinase switchesa / Schematics of the four tested recombinase switches based on site-specific recombinase Cre, FlpO and PhiC31. The switch constructs undergo site-specific recombination in order to express constitutively-driven reporter gene through various strategies (see also Supplementary Fig.1). Triangles indicate recombinase recognition sites. b / Maximal switch response (yellow bars) and corresponding efficiency (blue bars) of each switch. The efficiency is calculated as the ratio between recombinase-triggered reporter expression level and the expression level of pre-recombined control reporter in the presence of the same recombinase amount. Each bar represents mean±SD from three biological replicates measured 48 hours after transfection by flow cytometry (see also Supplementary Fig. 1). Plasmid amounts are provided in Supplementary Table 1. c / Schematics of the experimental setup for testing regulated recombinase activity. A CMV-driven transactivator is co-transfected with a recombinase driven by an appropriate inducible promoter and a recombinase-triggered switch. d / Top: activator-promoter combinations. Bottom: bar charts comparing the basal switch response (“−”, no activator) and the maximal response observed with different transactivators (“+”) (see also Supplementary Fig. 2). Each bar represents mean±SD of three biological replicates measured 48 hours after transfection by flow cytometry. Plasmid amounts are provided in Supplementary Table 2 and quantitative readouts of key samples in Supplementary Table 3.
Mentions: We first developed the irreversible knot that requires efficient recombination combined with irreversible genetic switch, and tight control of the recombinase by the transactivator to prevent uncontrolled switching and resulting output expression. We tested iCre42, FlpO43 and PhiC31o43 enzymes with several irreversible switches, including PhiC31-inversion44, Cre-excision45, Cre-Flex46 and FlpO-Flex47 (Fig. 2a, Supplementary Fig. 1a,b). We measured switches’ response to increasing dose of constitutively-expressed cognate recombinase and compared them to pre-recombined positive controls to estimate switching efficiency (Supplementary Fig. 1c, Supplementary Table 1, Supplementary Note 1). We found that all switches responded strongly to catalytic recombinase amounts. However only Cre-Flex and FlpO-Flex showed high absolute response and efficiency, and were retained for circuit development (Fig. 2b).

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