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Publications - Cell and Metabolic Biology

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Publications

Brückner, K.; Schäfer, P.; Weber, E.; Grützner, R.; Marillonnet, S.; Tissier, A.; A library of synthetic transcription activator-like effector-activated promoters for coordinated orthogonal gene expression in plants Plant J. 82, 707-716, (2015) DOI: 10.1111/tpj.12843

A library of synthetic promoters containing the binding site of a single designer transcription activator‐like effector (dTALE) was constructed. The promoters contain a constant sequence, consisting of an 18‐base long dTALE‐binding site and a TATA box, flanked by degenerate sequences of 49 bases downstream and 19 bases upstream. Forty‐three of these promoters were sequenced and tested in transient assays in Nicotiana benthamiana using a GUS reporter gene. The strength of expression of the promoters ranged from around 5% to almost 100% of the viral 35S promoter activity. We then demonstrated the utility of these promoters for metabolic engineering by transiently expressing three genes for the production of a plant diterpenoid in N. benthamiana. The simplicity of the promoter structure shows great promise for the development of genetic circuits, with wide potential applications in plant synthetic biology and metabolic engineering.
Publications

Werner, S.; Engler, C.; Weber, E.; Gruetzner, R.; Marillonnet, S.; Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system Bioengineered 3, 38-43, (2012) DOI: 10.4161/bbug.3.1.18223

Recent progress in the field of synthetic biology has led to the creation of cells containing synthetic genomes. Although these first synthetic organisms contained copies of natural genomes, future work will be directed toward engineering of organisms with modified genomes and novel phenotypes. Much work, however, remains to be done to be able to routinely engineer novel biological functions. As a tool that will be useful for such purpose, we have recently developed a modular cloning system (MoClo) that allows high throughput assembly of multiple genetic elements. We present here new features of this cloning system that allow to increase the speed of assembly of multigene constructs. As an example, 68 DNA fragments encoding basic genetic elements were assembled using three one-pot cloning steps, resulting in a 50 kb construct containing 17 eukaryotic transcription units. This cloning system should be useful for generating the multiple construct variants that will be required for developing gene networks encoding novel functions, and fine-tuning the expression levels of the various genes involved.
Publications

Weber, E.; Gruetzner, R.; Werner, S.; Engler, C.; Marillonnet, S.; Assembly of Designer TAL Effectors by Golden Gate Cloning PLOS ONE 6, e19722, (2011) DOI: 10.1371/journal.pone.0019722

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.
Publications

Weber, E.; Engler, C.; Gruetzner, R.; Werner, S.; Marillonnet, S.; A Modular Cloning System for Standardized Assembly of Multigene Constructs PLOS ONE 6, e16765, (2011) DOI: 10.1371/journal.pone.0016765

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.
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