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Rule-Based Design of Plant Expression Vectors Using GenoCAD.

Coll A, Wilson ML, Gruden K, Peccoud J - PLoS ONE (2015)

Bottom Line: It includes a library of plant biological parts organized in structural categories and a set of rules describing how to assemble these parts into large constructs.Rules developed here are organized and divided into three main subsections according to the aim of the final construct: protein localization studies, promoter analysis and protein-protein interaction experiments.The GenoCAD plant grammar guides the user through the design while allowing users to customize vectors according to their needs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia.

ABSTRACT
Plant synthetic biology requires software tools to assist on the design of complex multi-genic expression plasmids. Here a vector design strategy to express genes in plants is formalized and implemented as a grammar in GenoCAD, a Computer-Aided Design software for synthetic biology. It includes a library of plant biological parts organized in structural categories and a set of rules describing how to assemble these parts into large constructs. Rules developed here are organized and divided into three main subsections according to the aim of the final construct: protein localization studies, promoter analysis and protein-protein interaction experiments. The GenoCAD plant grammar guides the user through the design while allowing users to customize vectors according to their needs. Therefore the plant grammar implemented in GenoCAD will help plant biologists take advantage of methods from synthetic biology to design expression vectors supporting their research projects.

No MeSH data available.


Related in: MedlinePlus

Example of three different designs obtained following ppi route.A) bifc route. B) coip route using MYC and HA as epitope tags. C) coip route using GFP and HIS as epitope tags.
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pone.0132502.g003: Example of three different designs obtained following ppi route.A) bifc route. B) coip route using MYC and HA as epitope tags. C) coip route using GFP and HIS as epitope tags.

Mentions: In BiFC approaches, two potential interacting proteins are fused to two fragments of a fluorescent protein, and identification of interactions is based on the reconstitution of the split fluorescent tag [31]. In the CoIP assay, the protein complex is precipitated with an immobilized antibody against one of the proteins studied, and the interacting partner is further confirmed by Western Blot. Usually antibodies against tagged fusion proteins are used [32]. According to the principle of these methodologies, the most important specificity of both studies is that 2 vectors have to be designed in parallel to allow proper experimental design. A split fluorescent protein is required in vectors for BiFC studies, and 2 different epitope tags in the case of the CoIP approach. To satisfy these requirements, the ppi route implemented in our grammar gives the users two options i.e. bifc and coip route (S2 Table). In the first case (Fig 3A), the rule defines that the design involves 2 plasmids, the first one containing the C-terminal of a half-fluorescent protein fused to the target protein (GFPC), and the second one containing the N-terminal of the same fluorescent protein (GFPN). Epitope tags and/or linker domains can also be added at both sides of the fluorescent protein (rules tfpc, fpct, lfpc, fpcl, tfpn, fpnt, lfpn, fpnl). On the other hand, the coip rule defines that the design involves 2 plasmids containing two different epitope tags fused to the potential interacting proteins. According to the most commonly used tags in plant molecular biology, two-pair combinations of epitope tags are included in the grammar i.e. c-myc (MYC)/Human influenza hemagglutinin (HA) and GFP/poly-histidine (HIS) tag. Specifically, rule myha defines that the first plasmid contains a cassette, which includes a gene fused to MYC epitope tag (GMY), and the second plasmid contains the gene fused to the HA epitope (GHA) (Fig 3B). In the same way, rule gfhi forces the user to design the first plasmid with GFP fused to the gene (GGF), and the second plasmid with a gene fused to HIS tag (GHI) (Fig 3C). As in the case of localization studies, GEN category allow the user to add epitope tags and linkers at both sides of the protein under study, giving again high flexibility to the design.


Rule-Based Design of Plant Expression Vectors Using GenoCAD.

Coll A, Wilson ML, Gruden K, Peccoud J - PLoS ONE (2015)

Example of three different designs obtained following ppi route.A) bifc route. B) coip route using MYC and HA as epitope tags. C) coip route using GFP and HIS as epitope tags.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492961&req=5

pone.0132502.g003: Example of three different designs obtained following ppi route.A) bifc route. B) coip route using MYC and HA as epitope tags. C) coip route using GFP and HIS as epitope tags.
Mentions: In BiFC approaches, two potential interacting proteins are fused to two fragments of a fluorescent protein, and identification of interactions is based on the reconstitution of the split fluorescent tag [31]. In the CoIP assay, the protein complex is precipitated with an immobilized antibody against one of the proteins studied, and the interacting partner is further confirmed by Western Blot. Usually antibodies against tagged fusion proteins are used [32]. According to the principle of these methodologies, the most important specificity of both studies is that 2 vectors have to be designed in parallel to allow proper experimental design. A split fluorescent protein is required in vectors for BiFC studies, and 2 different epitope tags in the case of the CoIP approach. To satisfy these requirements, the ppi route implemented in our grammar gives the users two options i.e. bifc and coip route (S2 Table). In the first case (Fig 3A), the rule defines that the design involves 2 plasmids, the first one containing the C-terminal of a half-fluorescent protein fused to the target protein (GFPC), and the second one containing the N-terminal of the same fluorescent protein (GFPN). Epitope tags and/or linker domains can also be added at both sides of the fluorescent protein (rules tfpc, fpct, lfpc, fpcl, tfpn, fpnt, lfpn, fpnl). On the other hand, the coip rule defines that the design involves 2 plasmids containing two different epitope tags fused to the potential interacting proteins. According to the most commonly used tags in plant molecular biology, two-pair combinations of epitope tags are included in the grammar i.e. c-myc (MYC)/Human influenza hemagglutinin (HA) and GFP/poly-histidine (HIS) tag. Specifically, rule myha defines that the first plasmid contains a cassette, which includes a gene fused to MYC epitope tag (GMY), and the second plasmid contains the gene fused to the HA epitope (GHA) (Fig 3B). In the same way, rule gfhi forces the user to design the first plasmid with GFP fused to the gene (GGF), and the second plasmid with a gene fused to HIS tag (GHI) (Fig 3C). As in the case of localization studies, GEN category allow the user to add epitope tags and linkers at both sides of the protein under study, giving again high flexibility to the design.

Bottom Line: It includes a library of plant biological parts organized in structural categories and a set of rules describing how to assemble these parts into large constructs.Rules developed here are organized and divided into three main subsections according to the aim of the final construct: protein localization studies, promoter analysis and protein-protein interaction experiments.The GenoCAD plant grammar guides the user through the design while allowing users to customize vectors according to their needs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia.

ABSTRACT
Plant synthetic biology requires software tools to assist on the design of complex multi-genic expression plasmids. Here a vector design strategy to express genes in plants is formalized and implemented as a grammar in GenoCAD, a Computer-Aided Design software for synthetic biology. It includes a library of plant biological parts organized in structural categories and a set of rules describing how to assemble these parts into large constructs. Rules developed here are organized and divided into three main subsections according to the aim of the final construct: protein localization studies, promoter analysis and protein-protein interaction experiments. The GenoCAD plant grammar guides the user through the design while allowing users to customize vectors according to their needs. Therefore the plant grammar implemented in GenoCAD will help plant biologists take advantage of methods from synthetic biology to design expression vectors supporting their research projects.

No MeSH data available.


Related in: MedlinePlus