RESUMO
Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. Here, a toolkit containing further modules for the novel DNA assembly standards was developed. Intended for use with Modular Cloning, most modules are also compatible with GoldenBraid. Firstly, a collection of approximately 80 additional phytobricks is provided, comprising e.g. modules for inducible expression systems, promoters or epitope tags. Furthermore, DNA modules were developed for connecting Modular Cloning and Gateway cloning, either for toggling between systems or for standardized Gateway destination vector assembly. Finally, first instances of a "peripheral infrastructure" around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. The presented material will further enhance versatility of hierarchical DNA assembly strategies.
Assuntos
Clonagem Molecular/métodos , Engenharia Genética/métodos , Vetores Genéticos/química , Nicotiana/genética , Proteínas de Plantas/genética , Plasmídeos/química , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Calmodulina/genética , Calmodulina/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Vetores Genéticos/metabolismo , Fases de Leitura Aberta , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Nicotiana/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismoRESUMO
Plant Synthetic Biology requires robust and efficient methods for assembling multigene constructs. Golden Gate cloning provides a precision module-based cloning technique for facile assembly of multiple genes in one construct. We present here a versatile resource for plant biologists comprising a set of cloning vectors and 96 standardized parts to enable Golden Gate construction of multigene constructs for plant transformation. Parts include promoters, untranslated sequences, reporters, antigenic tags, localization signals, selectable markers, and terminators. The comparative performance of parts in the model plant Nicotiana benthamiana is discussed.
Assuntos
Clonagem Molecular/métodos , Engenharia Genética/métodos , Vetores Genéticos/genética , Biologia Sintética/métodos , Agrobacterium tumefaciens/genética , Modelos Genéticos , Nicotiana/genéticaRESUMO
Generation of customized DNA binding domains targeting unique sequences in complex genomes is crucial for many biotechnological applications. The recently described DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas consists of a series of repeats arranged in tandem, each repeat binding a nucleotide of the target sequence. We present here a strategy for engineering of TALE proteins with novel DNA binding specificities based on the 17.5 repeat-containing AvrBs3 TALE as a scaffold. For each of the 17 full repeats, four module types were generated, each with a distinct base preference. Using this set of 68 repeat modules, recognition domains for any 17 nucleotide DNA target sequence of choice can be constructed by assembling selected modules in a defined linear order. Assembly is performed in two successive one-pot cloning steps using the Golden Gate cloning method that allows seamless fusion of multiple DNA fragments. Applying this strategy, we assembled designer TALEs with new target specificities and tested their function in vivo.
Assuntos
Clonagem Molecular , Proteínas de Ligação a DNA/genética , DNA/metabolismo , Transativadores/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Vetores Genéticos , Genoma de Planta , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ligação Proteica , Engenharia de Proteínas , Sequências Repetitivas de Ácido Nucleico/genética , Homologia de Sequência do Ácido Nucleico , Nicotiana/genética , Transativadores/química , Transativadores/metabolismo , Fatores de Transcrição/genéticaRESUMO
The field of synthetic biology promises to revolutionize biotechnology through the design of organisms with novel phenotypes useful for medicine, agriculture and industry. However, a limiting factor is the ability of current methods to assemble complex DNA molecules encoding multiple genetic elements in various predefined arrangements. We present here a hierarchical modular cloning system that allows the creation at will and with high efficiency of any eukaryotic multigene construct, starting from libraries of defined and validated basic modules containing regulatory and coding sequences. This system is based on the ability of type IIS restriction enzymes to assemble multiple DNA fragments in a defined linear order. We constructed a 33 kb DNA molecule containing 11 transcription units made from 44 individual basic modules in only three successive cloning steps. This modular cloning (MoClo) system can be readily automated and will be extremely useful for applications such as gene stacking and metabolic engineering.