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Post-translational control of genetic circuits using Potyvirus proteases.

ABSTRACT: Genetic engineering projects often require control over when a protein is degraded. To this end, we use a fusion between a degron and an inactivating peptide that can be added to the N-terminus of a protein. When the corresponding protease is expressed, it cleaves the peptide and the protein is degraded. Three protease:cleavage site pairs from Potyvirus are shown to be orthogonal and active in exposing degrons, releasing inhibitory domains and cleaving polyproteins. This toolbox is applied to the design of genetic circuits as a means to control regulator activity and degradation. First, we demonstrate that a gate can be constructed by constitutively expressing an inactivated repressor and having an input promoter drive the expression of the protease. It is also shown that the proteolytic release of an inhibitory domain can improve the dynamic range of a transcriptional gate (200-fold repression). Next, we design polyproteins containing multiple repressors and show that their cleavage can be used to control multiple outputs. Finally, we demonstrate that the dynamic range of an output can be improved (8-fold to 190-fold) with the addition of a protease-cleaved degron. Thus, controllable proteolysis offers a powerful tool for modulating and expanding the function of synthetic gene circuits.

SUBMITTER: Fernandez-Rodriguez J

PROVIDER: S-EPMC5291274 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Post-translational control of genetic circuits using Potyvirus proteases.

Fernandez-Rodriguez Jesus J Fernandez-Rodriguez Jesus J Voigt Christopher A CA

Nucleic acids research 20160613 13

Genetic engineering projects often require control over when a protein is degraded. To this end, we use a fusion between a degron and an inactivating peptide that can be added to the N-terminus of a protein. When the corresponding protease is expressed, it cleaves the peptide and the protein is degraded. Three protease:cleavage site pairs from Potyvirus are shown to be orthogonal and active in exposing degrons, releasing inhibitory domains and cleaving polyproteins. This toolbox is applied to th ...[more]

PMID: 27298256

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Post-translational control of genetic circuits using Potyvirus proteases.

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Post-translational control of genetic circuits using Potyvirus proteases.

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