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Publications - Cell and Metabolic Biology

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Printed publications

Grützner, R.; König, K.; Horn, C.; Engler, C.; Laub, A.; Vogt, T.; Marillonnet, S.; A transient expression tool box for anthocyanin biosynthesis in Nicotiana benthamiana Plant Biotechnol. J. (2023) DOI: 10.1111/pbi.14261

Transient expression in Nicotiana benthamiana offers a robust platform for the rapid production of complex secondary metabolites. It has proven highly effective in helping identify genes associated with pathways responsible for synthesizing various valuable natural compounds. While this approach has seen considerable success, it has yet to be applied to uncovering genes involved in anthocyanin biosynthetic pathways. This is because only a single anthocyanin, delphinidin 3‐O‐rutinoside, can be produced in N. benthamiana by activation of anthocyanin biosynthesis using transcription factors. The production of other anthocyanins would necessitate the suppression of certain endogenous flavonoid biosynthesis genes while transiently expressing others. In this work, we present a series of tools for the reconstitution of anthocyanin biosynthetic pathways in N. benthamiana leaves. These tools include constructs for the expression or silencing of anthocyanin biosynthetic genes and a mutant N. benthamiana line generated using CRISPR. By infiltration of defined sets of constructs, the basic anthocyanins pelargonidin 3‐O‐glucoside, cyanidin 3‐O‐glucoside and delphinidin 3‐O‐glucoside could be obtained in high amounts in a few days. Additionally, co‐infiltration of supplementary pathway genes enabled the synthesis of more complex anthocyanins. These tools should be useful to identify genes involved in the biosynthesis of complex anthocyanins. They also make it possible to produce novel anthocyanins not found in nature. As an example, we reconstituted the pathway for biosynthesis of Arabidopsis anthocyanin A5, a cyanidin derivative and achieved the biosynthesis of the pelargonidin and delphinidin variants of A5, pelargonidin A5 and delphinidin A5.
Publications

Engler, C.; Youles, M.; Gruetzner, R.; Ehnert, T.-M.; Werner, S.; Jones, J. D. G.; Patron, N. J.; Marillonnet, S.; A Golden Gate Modular Cloning Toolbox for Plants ACS Synth. Biol. 3, 839-843, (2014) DOI: 10.1021/sb4001504

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.
Books and chapters

Engler, C.; Marillonnet, S.; Golden Gate Cloning (Valla, S. & Lale, R., eds.). Methods Mol. Biol. 1116, 119-131, (2014) ISBN: 978-1-62703-764-8 DOI: 10.1007/978-1-62703-764-8_9

DNA assembly methods are essential tools for biological research and biotechnology. Therefore various methods have been developed to clone DNA fragments of interest. Conventional methods usually require several cloning steps to generate a construct of interest. At each step, a single DNA fragment is transferred from a donor plasmid or PCR product to a recipient vector. In the past few years, a number of methods have been developed to facilitate and speed up this process. One of these methods, Golden Gate cloning, allows assembling up to nine fragments at a time in a recipient plasmid. Cloning is performed by pipetting in a single tube all plasmid donors, the recipient vector, a type IIS restriction enzyme and ligase, and incubating the mix in a thermal cycler. Despite the simplicity of the cloning procedure, the majority of clones obtained after transformation contain the expected construct. Using Golden Gate cloning however requires the use of carefully designed donor and recipient plasmids. We provide here a protocol describing how to design these plasmids and also describe the conditions necessary to perform the assembly reaction.
Books and chapters

Engler, C.; Marillonnet, S.; Combinatorial DNA Assembly Using Golden Gate Cloning (Polizzi, K. M. & Kontoravdi, C., eds.). Methods Mol. Biol. 1073, 141-156, (2013) ISBN: 978-1-62703-625-2 DOI: 10.1007/978-1-62703-625-2_12

A basic requirement for synthetic biology is the availability of efficient DNA assembly methods. We have previously reported the development of Golden Gate cloning, a method that allows parallel assembly of multiple DNA fragments in a one-tube reaction. Golden Gate cloning can be used for different levels of construct assembly: from gene fragments to complete gene coding sequences, from basic genetic elements to full transcription units, and finally from transcription units to multigene constructs. We provide here a protocol for DNA assembly using Golden Gate cloning, taking as an example the level of assembly of gene fragments to complete coding sequences, a level of cloning that can be used to perform DNA shuffling. Such protocol requires the following steps: (1) selecting fusion sites within parental sequences (sites at which parental sequences will be recombined), (2) amplifying all DNA fragments by PCR to add flanking restriction sites, (3) cloning the amplified fragments in intermediate constructs, and (4) assembling all or selected sets of intermediate constructs in a compatible recipient vector using a one-pot restriction-ligation.
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.
Publications

Thieme, F.; Engler, C.; Kandzia, R.; Marillonnet, S.; Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes PLOS ONE 6, e20556, (2011) DOI: 10.1371/journal.pone.0020556

We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplification of the insert. The other side of the linearized cloning vector has homology with a sequence present in the insert, but nested and non-overlapping with the gene-specific primer used for amplification. Since only specific products contain this sequence, but none of the non-specific products, only specific products can be cloned. Cloning is performed using a one-step reaction that only requires incubation for 10 minutes at room temperature in the presence of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. The reaction mix is then directly transformed into E. coli where the annealed vector-insert complex is repaired and ligated. We have tested this method, which we call quick and clean cloning (QC cloning), for cloning of the variable regions of immunoglobulins expressed in non-Hodgkin lymphoma tumor samples. This method can also be applied to identify the flanking sequence of DNA elements such as T-DNA or transposon insertions, or be used for cloning of any PCR product with high specificity.
Books and chapters

Engler, C.; Marillonnet, S.; Generation of Families of Construct Variants Using Golden Gate Shuffling (Lu, C. et al., eds.). Methods Mol. Biol. 729, 167-181, (2011) ISBN: 978-1-61779-065-2 DOI: 10.1007/978-1-61779-065-2_11

Current standard cloning methods based on the use of restriction enzymes and ligase are very versatile, but are not well suited for high-throughput cloning projects or for assembly of many DNA fragments from several parental plasmids in a single step. We have previously reported the development of an efficient cloning method based on the use of type IIs restriction enzymes and restriction–ligation. Such method allows seamless assembly of multiple fragments from several parental plasmids with high efficiency, and also allows performing DNA shuffling if fragments prepared from several homologous genes are assembled together in a single restriction–ligation. Such protocol, called Golden Gate shuffling, requires performing the following steps: (1) sequences from several homologous genes are aligned, and recombination sites defined on conserved sequences; (2) modules defined by the position of these recombination sites are amplified by PCR with primers designed to equip them with flanking BsaI sites; (3) the amplified fragments are cloned as intermediate constructs and sequenced; and (4) finally, the intermediate modules are assembled together in a compatible recipient vector in a one-pot restriction–ligation. Depending on the needs of the user, and because of the high cloning efficiency, the resulting constructs can either be screened and analyzed individually, or, if required in larger numbers, directly used in functional screens to detect improved protein variants.
Publications

Engler, C.; Gruetzner, R.; Kandzia, R.; Marillonnet, S.; Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes PLOS ONE 4, e5553, (2009) DOI: 10.1371/journal.pone.0005553

We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.
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