Dataset Information

COLORFUL-Circuit: A Platform for Rapid Multigene Assembly, Delivery, and Expression in Plants.

ABSTRACT: Advancing basic and applied plant research requires the continuous innovative development of the available technology toolbox. Essential components of this toolbox are methods that simplify the assembly, delivery, and expression of multiple transgenes of interest. To allow simultaneous and directional multigene assembly on the same plant transformation vector, several strategies based on overlapping sequences or restriction enzymes have recently been developed. However, the assembly of homologous and repetitive DNA sequences can be inefficient and the frequent occurrence of target sequences recognized by commonly used restriction enzymes can be a limiting factor. Here, we noted that recognition sites for the restriction enzyme SfiI are rarely occurring in plant genomes. This fact was exploited to establish a multigene assembly system called "COLORFUL-Circuit." To this end, we developed a set of binary vectors which provide a flexible and cost efficient cloning platform. The gene expression cassettes in our system are flanked with unique SfiI sites, which allow simultaneous multi-gene cassette assembly in a hosting binary vector. We used COLORFUL-Circuit to transiently and stably express up to four fluorescent organelle markers in addition to a selectable marker and analyzed the impact of assembly design on coexpression efficiency. Finally, we demonstrate the utility of our optimized "COLORFUL-Circuit" system in an exemplary case study, in which we monitored simultaneously the subcellular behavior of multiple organelles in a biotrophic plant-microbe interaction by Confocal Laser Scanning Microscopy.

SUBMITTER: Ghareeb H

PROVIDER: S-EPMC4772762 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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COLORFUL-Circuit: A Platform for Rapid Multigene Assembly, Delivery, and Expression in Plants.

Ghareeb Hassan H Laukamm Sabine S Lipka Volker V

Frontiers in plant science 20160301

Advancing basic and applied plant research requires the continuous innovative development of the available technology toolbox. Essential components of this toolbox are methods that simplify the assembly, delivery, and expression of multiple transgenes of interest. To allow simultaneous and directional multigene assembly on the same plant transformation vector, several strategies based on overlapping sequences or restriction enzymes have recently been developed. However, the assembly of homologou ...[more]

PMID: 26973687

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Project description:Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scar-free and sequence-independent multigene assembly strategy AssemblX, based on overlap-depended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science.

Dataset Information

COLORFUL-Circuit: A Platform for Rapid Multigene Assembly, Delivery, and Expression in Plants.

Publications

COLORFUL-Circuit: A Platform for Rapid Multigene Assembly, Delivery, and Expression in Plants.

Similar Datasets

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