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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 operation in different cell linesa / Schematics of V2.1 circuits and corresponding controls specific to this set of experiments. The constitutive On and Off controls are used to measure the range of expression that is specific to each cell line. TFF5 is a control sequence that is not known to be targeted by any known human miRNA. b / Citrine (top) and Cerulean (bottom) readouts of the complete circuits and relevant controls compared across cell lines. Shown are mean±SD of three independent biological replicates measured 48 hours after transfection by flow cytometry. Two-sided unpaired t-tests were performed to compare the differential output expression of the Circuit sample in HEK293 (Off state) compared to HeLa and Huh-7 (On states). P-values < 0.0001 are indicated. Transfection setup is given in Supplementary Table 14, quantitative values in Supplementary Table 15 and scatter plots in Supplementary Fig. 11.
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Figure 8: Circuit operation in different cell linesa / Schematics of V2.1 circuits and corresponding controls specific to this set of experiments. The constitutive On and Off controls are used to measure the range of expression that is specific to each cell line. TFF5 is a control sequence that is not known to be targeted by any known human miRNA. b / Citrine (top) and Cerulean (bottom) readouts of the complete circuits and relevant controls compared across cell lines. Shown are mean±SD of three independent biological replicates measured 48 hours after transfection by flow cytometry. Two-sided unpaired t-tests were performed to compare the differential output expression of the Circuit sample in HEK293 (Off state) compared to HeLa and Huh-7 (On states). P-values < 0.0001 are indicated. Transfection setup is given in Supplementary Table 14, quantitative values in Supplementary Table 15 and scatter plots in Supplementary Fig. 11.

Mentions: As HeLa and HuH-7 have lower transfection efficiency, we utilized an earlier finding that placing SV40-iCre on the same backbone with the fan-in module output increases the inversion efficiency (Lapique et al, manuscript in revision). Thus we combined SV40-iCre and Cerulean-2A-PIT2 cassettes on the same plasmid and adjusted upwards the amount of this construct to reach a higher On state. The circuits with this modification were dubbed V2.1-FlpO and V2.1-Rev (Fig. 8a). Both of them responded correctly to their endogenous miRNA inputs in HeLa, HuH-7 and HEK293 cells. We also measured a number of control circuits to validate our findings (Fig. 8b, Supplementary Fig. 11, Supplementary Tables 14, 15). In the reversible case, the trends were fully consistent with expectation. The control ΔS21 generated similar readouts to the On circuit in HeLa and HEK293, and a reduced readout in HuH-7 due to the expression of miR-142a (which acts on Cerulean-2A-PIT2). Both negative controls --- “Off” and ΔPIT2 --- were very low, as expected. The complete circuit was On in HeLa and HuH-7 with Cerulean readout reduced relative to respective ΔS21 readout and with Citrine output tracking the Cerulean. In the irreversible case, full circuit readouts were slightly higher compared to ΔS21, which is contrary to expectation but can be explained by experimental variability. More interestingly, the differences in Citrine output did not track respective differences in Cerulean in HeLa and HuH-7 cells in both the circuits and the controls. A simple explanation is that the recombination efficiency and/or speed are higher in HuH-7 cells than in HeLa, thus the same amount of PIT2/Cre generates different outputs. Nevertheless, qualitative circuit function was consistent with expectation without requiring overly extensive optimization. Thus our architecture is applicable to different scenarios and is not strictly limited to the chassis cell line where it has been originally established.


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

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

Circuit operation in different cell linesa / Schematics of V2.1 circuits and corresponding controls specific to this set of experiments. The constitutive On and Off controls are used to measure the range of expression that is specific to each cell line. TFF5 is a control sequence that is not known to be targeted by any known human miRNA. b / Citrine (top) and Cerulean (bottom) readouts of the complete circuits and relevant controls compared across cell lines. Shown are mean±SD of three independent biological replicates measured 48 hours after transfection by flow cytometry. Two-sided unpaired t-tests were performed to compare the differential output expression of the Circuit sample in HEK293 (Off state) compared to HeLa and Huh-7 (On states). P-values < 0.0001 are indicated. Transfection setup is given in Supplementary Table 14, quantitative values in Supplementary Table 15 and scatter plots in Supplementary Fig. 11.
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Figure 8: Circuit operation in different cell linesa / Schematics of V2.1 circuits and corresponding controls specific to this set of experiments. The constitutive On and Off controls are used to measure the range of expression that is specific to each cell line. TFF5 is a control sequence that is not known to be targeted by any known human miRNA. b / Citrine (top) and Cerulean (bottom) readouts of the complete circuits and relevant controls compared across cell lines. Shown are mean±SD of three independent biological replicates measured 48 hours after transfection by flow cytometry. Two-sided unpaired t-tests were performed to compare the differential output expression of the Circuit sample in HEK293 (Off state) compared to HeLa and Huh-7 (On states). P-values < 0.0001 are indicated. Transfection setup is given in Supplementary Table 14, quantitative values in Supplementary Table 15 and scatter plots in Supplementary Fig. 11.
Mentions: As HeLa and HuH-7 have lower transfection efficiency, we utilized an earlier finding that placing SV40-iCre on the same backbone with the fan-in module output increases the inversion efficiency (Lapique et al, manuscript in revision). Thus we combined SV40-iCre and Cerulean-2A-PIT2 cassettes on the same plasmid and adjusted upwards the amount of this construct to reach a higher On state. The circuits with this modification were dubbed V2.1-FlpO and V2.1-Rev (Fig. 8a). Both of them responded correctly to their endogenous miRNA inputs in HeLa, HuH-7 and HEK293 cells. We also measured a number of control circuits to validate our findings (Fig. 8b, Supplementary Fig. 11, Supplementary Tables 14, 15). In the reversible case, the trends were fully consistent with expectation. The control ΔS21 generated similar readouts to the On circuit in HeLa and HEK293, and a reduced readout in HuH-7 due to the expression of miR-142a (which acts on Cerulean-2A-PIT2). Both negative controls --- “Off” and ΔPIT2 --- were very low, as expected. The complete circuit was On in HeLa and HuH-7 with Cerulean readout reduced relative to respective ΔS21 readout and with Citrine output tracking the Cerulean. In the irreversible case, full circuit readouts were slightly higher compared to ΔS21, which is contrary to expectation but can be explained by experimental variability. More interestingly, the differences in Citrine output did not track respective differences in Cerulean in HeLa and HuH-7 cells in both the circuits and the controls. A simple explanation is that the recombination efficiency and/or speed are higher in HuH-7 cells than in HeLa, thus the same amount of PIT2/Cre generates different outputs. Nevertheless, qualitative circuit function was consistent with expectation without requiring overly extensive optimization. Thus our architecture is applicable to different scenarios and is not strictly limited to the chassis cell line where it has been originally established.

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