Dataset Information

Design of orthogonal genetic switches based on a crosstalk map of ?s, anti-?s, and promoters.

ABSTRACT: Cells react to their environment through gene regulatory networks. Network integrity requires minimization of undesired crosstalk between their biomolecules. Similar constraints also limit the use of regulators when building synthetic circuits for engineering applications. Here, we mapped the promoter specificities of extracytoplasmic function (ECF) ?s as well as the specificity of their interaction with anti-?s. DNA synthesis was used to build 86 ECF ?s (two from every subgroup), their promoters, and 62 anti-?s identified from the genomes of diverse bacteria. A subset of 20 ?s and promoters were found to be highly orthogonal to each other. This set can be increased by combining the -35 and -10 binding domains from different subgroups to build chimeras that target sequences unrepresented in any subgroup. The orthogonal ?s, anti-?s, and promoters were used to build synthetic genetic switches in Escherichia coli. This represents a genome-scale resource of the properties of ECF ?s and a resource for synthetic biology, where this set of well-characterized regulatory parts will enable the construction of sophisticated gene expression programs.

SUBMITTER: Rhodius VA

PROVIDER: S-EPMC3817407 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters.

Rhodius Virgil A VA Segall-Shapiro Thomas H TH Sharon Brian D BD Ghodasara Amar A Orlova Ekaterina E Tabakh Hannah H Burkhardt David H DH Clancy Kevin K Peterson Todd C TC Gross Carol A CA Voigt Christopher A CA

Molecular systems biology 20131029

Cells react to their environment through gene regulatory networks. Network integrity requires minimization of undesired crosstalk between their biomolecules. Similar constraints also limit the use of regulators when building synthetic circuits for engineering applications. Here, we mapped the promoter specificities of extracytoplasmic function (ECF) σs as well as the specificity of their interaction with anti-σs. DNA synthesis was used to build 86 ECF σs (two from every subgroup), their promoter ...[more]

PMID: 24169405

Similar Datasets

Project description:BackgroundThe construction and application of synthetic genetic circuits is frequently improved if gene expression can be orthogonally controlled, relative to the host. In plants, orthogonality can be achieved via the use of CRISPR-based transcription factors that are programmed to act on natural or synthetic promoters. The construction of complex gene circuits can require multiple, orthogonal regulatory interactions, and this in turn requires that the full programmability of CRISPR elements be adapted to non-natural and non-standard promoters that have few constraints on their design. Therefore, we have developed synthetic promoter elements in which regions upstream of the minimal 35S CaMV promoter are designed from scratch to interact via programmed gRNAs with dCas9 fusions that allow activation of gene expression.ResultsA panel of three, mutually orthogonal promoters that can be acted on by artificial gRNAs bound by CRISPR regulators were designed. Guide RNA expression targeting these promoters was in turn controlled by either Pol III (U6) or ethylene-inducible Pol II promoters, implementing for the first time a fully artificial Orthogonal Control System (OCS). Following demonstration of the complete orthogonality of the designs, the OCS was tied to cellular metabolism by putting gRNA expression under the control of an endogenous plant signaling molecule, ethylene. The ability to form complex circuitry was demonstrated via the ethylene-driven, ratiometric expression of fluorescent proteins in single plants.ConclusionsThe design of synthetic promoters is highly generalizable to large tracts of sequence space, allowing Orthogonal Control Systems of increasing complexity to potentially be generated at will. The ability to tie in several different basal features of plant molecular biology (Pol II and Pol III promoters, ethylene regulation) to the OCS demonstrates multiple opportunities for engineering at the system level. Moreover, given the fungibility of the core 35S CaMV promoter elements, the derived synthetic promoters can potentially be utilized across a variety of plant species.

Dataset Information

Design of orthogonal genetic switches based on a crosstalk map of ?s, anti-?s, and promoters.

Publications

Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets