Limits...
Digital switching in a biosensor circuit via programmable timing of gene availability.

Lapique N, Benenson Y - Nat. Chem. Biol. (2014)

Bottom Line: Here we show that site-specific recombinases can rectify undesired effects by programmable timing of gene availability in multigene circuits.The new sensors display a dynamic range of up to 1,000-fold compared to 20-fold in the standard configuration.Our study opens new venues in gene circuit design via judicious temporal control of circuits' genetic makeup.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosystems Science and Engineering, Swiss Federal Institute of Technology Zurich (ETHZ), Basel, Switzerland.

ABSTRACT
Transient delivery of gene circuits is required in many potential applications of synthetic biology, yet the pre-steady-state processes that dominate this delivery route pose major challenges for robust circuit deployment. Here we show that site-specific recombinases can rectify undesired effects by programmable timing of gene availability in multigene circuits. We exemplify the concept with a proportional sensor for endogenous microRNA (miRNA) and show a marked reduction in its ground state leakage due to desynchronization of the circuit's repressor components and their repression target. The new sensors display a dynamic range of up to 1,000-fold compared to 20-fold in the standard configuration. We applied the approach to classify cell types on the basis of miRNA expression profile and measured >200-fold output differential between positively and negatively identified cells. We also showed major improvements in specificity with cytotoxic output. Our study opens new venues in gene circuit design via judicious temporal control of circuits' genetic makeup.

Show MeSH

Related in: MedlinePlus

Component contribution to sensor performanceCircuit diagrams of miR-FF4 mutant sensor (a), LacI mutant sensor (b) and their relative performance (c). Asterisks indicate mutated components. Error bars show ± standard deviation from at least three biological replicas.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4232471&req=5

Figure 4: Component contribution to sensor performanceCircuit diagrams of miR-FF4 mutant sensor (a), LacI mutant sensor (b) and their relative performance (c). Asterisks indicate mutated components. Error bars show ± standard deviation from at least three biological replicas.

Mentions: The cumulative evidence shown above strongly favored the hypothesis that the recombinase-induced delay enabled the double-inversion module to produce enough repressor molecules prior to the commencement of output expression, thus greatly reducing the leakage. Next we asked which molecular features of the double-inversion module contributed most to this phenomenon. The repressor is a combined transcriptional/post-transcriptional unit that uses LacI and an artificial spliced miRNA (miR-FF4), respectively. We already showed that siRNA-FF4 had very strong inhibitory effect of its own (Supplementary Fig. 6). We mutated either the LacI or the miR-FF4 component (Fig. 4a,b), confirmed their loss-of-function (Supplementary Fig. 9), and measured sensor performance in both standard and delayed configurations (Fig. 4c). The data showed that while LacI-miR-FF4 combination was the best performer, the LacI mutant still generated most of the total repression capacity via miR-FF4. In the delayed configuration it exhibited a dynamic range of more than two orders of magnitude, and likewise in the standard setting the effect of LacI removal was relatively minor. On the other hand, removing miR-FF4 had a major detrimental effect on both delayed and standard architectures. This indicates that an efficient repressor is a necessary prerequisite for high dynamic range in both standard and delayed configurations.


Digital switching in a biosensor circuit via programmable timing of gene availability.

Lapique N, Benenson Y - Nat. Chem. Biol. (2014)

Component contribution to sensor performanceCircuit diagrams of miR-FF4 mutant sensor (a), LacI mutant sensor (b) and their relative performance (c). Asterisks indicate mutated components. Error bars show ± standard deviation from at least three biological replicas.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4232471&req=5

Figure 4: Component contribution to sensor performanceCircuit diagrams of miR-FF4 mutant sensor (a), LacI mutant sensor (b) and their relative performance (c). Asterisks indicate mutated components. Error bars show ± standard deviation from at least three biological replicas.
Mentions: The cumulative evidence shown above strongly favored the hypothesis that the recombinase-induced delay enabled the double-inversion module to produce enough repressor molecules prior to the commencement of output expression, thus greatly reducing the leakage. Next we asked which molecular features of the double-inversion module contributed most to this phenomenon. The repressor is a combined transcriptional/post-transcriptional unit that uses LacI and an artificial spliced miRNA (miR-FF4), respectively. We already showed that siRNA-FF4 had very strong inhibitory effect of its own (Supplementary Fig. 6). We mutated either the LacI or the miR-FF4 component (Fig. 4a,b), confirmed their loss-of-function (Supplementary Fig. 9), and measured sensor performance in both standard and delayed configurations (Fig. 4c). The data showed that while LacI-miR-FF4 combination was the best performer, the LacI mutant still generated most of the total repression capacity via miR-FF4. In the delayed configuration it exhibited a dynamic range of more than two orders of magnitude, and likewise in the standard setting the effect of LacI removal was relatively minor. On the other hand, removing miR-FF4 had a major detrimental effect on both delayed and standard architectures. This indicates that an efficient repressor is a necessary prerequisite for high dynamic range in both standard and delayed configurations.

Bottom Line: Here we show that site-specific recombinases can rectify undesired effects by programmable timing of gene availability in multigene circuits.The new sensors display a dynamic range of up to 1,000-fold compared to 20-fold in the standard configuration.Our study opens new venues in gene circuit design via judicious temporal control of circuits' genetic makeup.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosystems Science and Engineering, Swiss Federal Institute of Technology Zurich (ETHZ), Basel, Switzerland.

ABSTRACT
Transient delivery of gene circuits is required in many potential applications of synthetic biology, yet the pre-steady-state processes that dominate this delivery route pose major challenges for robust circuit deployment. Here we show that site-specific recombinases can rectify undesired effects by programmable timing of gene availability in multigene circuits. We exemplify the concept with a proportional sensor for endogenous microRNA (miRNA) and show a marked reduction in its ground state leakage due to desynchronization of the circuit's repressor components and their repression target. The new sensors display a dynamic range of up to 1,000-fold compared to 20-fold in the standard configuration. We applied the approach to classify cell types on the basis of miRNA expression profile and measured >200-fold output differential between positively and negatively identified cells. We also showed major improvements in specificity with cytotoxic output. Our study opens new venues in gene circuit design via judicious temporal control of circuits' genetic makeup.

Show MeSH
Related in: MedlinePlus