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Publikationen - Stoffwechsel- und Zellbiologie

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Bücher und Buchkapitel

Marillonnet, S.; Werner, S.; Assembly of Multigene Constructs Using the Modular Cloning System MoClo (In: Chandran S., George K.). Methods Mol. Biol. 2205, 125-141, (2020) ISBN: 978-1-0716-0907-1 DOI: 10.1007/978-1-0716-0908-8_8

Modular cloning systems that rely on type IIS enzymes for DNA assembly have many advantages for complex pathway engineering. These systems are simple to use, efficient, and allow users to assemble multigene constructs by performing a series of one-pot assembly steps, starting from libraries of cloned and sequenced parts. The efficiency of these systems also facilitates the generation of libraries of construct variants. We describe here a protocol for assembly of multigene constructs using the Modular Cloning system MoClo. Making constructs using the MoClo system requires users to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. The assembly strategy is then defined following a set of standard rules. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.
Bücher und Buchkapitel

Hause, B.; Requena, N.; Detection of Arbuscular Mycorrhizal Fungal Gene Expression by In Situ Hybridization (Ferrol, N. & Lanfranco, L., eds.). Methods Mol. Biol. 2146, 185-196, (2020) ISBN: 978-1-0716-0603-2 DOI: 10.1007/978-1-0716-0603-2_14

The complexity of the obligate symbiotic interaction of arbuscular mycorrhizal (AM) fungi and their host roots requires sophisticated molecular methods. In particular, to capture the dynamic of the interaction, cell-specific methods for gene expression analysis are required. In situ hybridization is a technique that allows to determine the location of transcript accumulation within tissues, being of special interest for these fungi that cannot be genetically modified. The method requires proper fixation and embedding methods as well as specific probes for the hybridization allowing detection of specific transcripts. In this chapter, we present a method to prepare roots, which have established a symbiosis with an arbuscular mycorrhizal fungus for the detection of fungal transcripts. This includes chemical fixation, subsequent embedding in a suitable medium, sectioning and pretreatment of sections, the hybridization procedure itself, as well as the immunological detection of RNA-RNA hybrids.
Bücher und Buchkapitel

Grützner, R.; Marillonnet, S.; Generation of MoClo Standard Parts Using Golden Gate Cloning (In: Chandran S., George K.). Methods Mol. Biol. 2205, 107-123, (2020) ISBN: 978-1-0716-0907-1 DOI: 10.1007/978-1-0716-0908-8_7

Availability of efficient DNA assembly methods is a basic requirement for synthetic biology. A variety of modular cloning systems have been developed, based on Golden Gate cloning for DNA assembly, to enable users to assemble multigene constructs from libraries of standard parts using a series of successive one-pot assembly reactions. Standard parts contain the DNA sequence coding for a genetic element of interest such as a promoter, coding sequence or terminator. Standard parts for the modular cloning system MoClo must be flanked by two BsaI restriction sites and should not contain internal sequences for two type IIS restriction sites, BsaI and BpiI, and optionally for a third type IIS enzyme, BsmBI. We provide here a detailed protocol for cloning of basic parts. This protocol requires the following steps (1) defining the type of basic part that needs to be cloned, (2) designing primers for amplification, (3) performing PCR amplification, (4) cloning of the fragments using Golden Gate cloning, and finally (5) sequencing of the part. For large basic parts, it is preferable to first clone subparts as intermediate level −1 constructs. These subparts are sequenced individually and are then further assembled to make the final level 0 module.
Bücher und Buchkapitel

Marillonnet, S.; Werner, S.; Assembly of Complex Pathways Using Type IIs Restriction Enzymes (Santos, C. N. S. & Ajikumar, P. K., eds.). Methods Mol. Biol. 1927, 93-109, (2019) ISBN: 978-1-4939-9142-6 DOI: 10.1007/978-1-4939-9142-6_7

Efficient DNA assembly methods are essential tools for synthetic biology and metabolic engineering. Among several recently developed methods that allow assembly of multiple DNA fragments in a single step, DNA assembly using type IIS enzymes provides many advantages for complex pathway engineering. In particular, it provides the ability for the user to quickly assemble multigene constructs using a series of simple one-pot assembly steps starting from libraries of cloned and sequenced parts. We describe here a protocol for assembly of multigene constructs using the modular cloning system (MoClo). Making constructs using the MoClo system requires to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. Basic parts that are not yet available need to be made. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.
Bücher und Buchkapitel

Janik, K.; Stellmach, H.; Mittelberger, C.; Hause, B.; Characterization of Phytoplasmal Effector Protein Interaction with Proteinaceous Plant Host Targets Using Bimolecular Fluorescence Complementation (BiFC) (Musetti, R. & Pagliari, L., eds.). Methods Mol. Biol. 1875, 321-331, (2019) ISBN: 978-1-4939-8837-2 DOI: 10.1007/978-1-4939-8837-2_24

Elucidating the molecular mechanisms underlying plant disease development has become an important aspect of phytoplasma research in the last years. Especially unraveling the function of phytoplasma effector proteins has gained interesting insights into phytoplasma-host interaction at the molecular level. Here, we describe how to analyze and visualize the interaction of a phytoplasma effector with its proteinaceous host partner using bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana mesophyll protoplasts. The protocol comprises a description of how to isolate protoplasts from leaves and how to transform these protoplasts with BiFC expression vectors containing the phytoplasma effector and the host interaction partner, respectively. If an interaction occurs, a fluorescent YFP-complex is reconstituted in the protoplast, which can be visualized using fluorescence microscopy.
Bücher und Buchkapitel

Schreiber, T.; Tissier, A.; Generation of dTALEs and Libraries of Synthetic TALE-Activated Promoters for Engineering of Gene Regulatory Networks in Plants (Kaufmann, K. & Mueller-Roeber, B., eds.). Methods Mol. Biol. 1629, 185-204, (2017) ISBN: 978-1-49397-125-1 DOI: 10.1007/978-1-4939-7125-1_13

Transcription factors with programmable DNA-binding specificity constitute valuable tools for the design of orthogonal gene regulatory networks for synthetic biology. Transcription activator-like effectors (TALEs), as natural transcription regulators, were used to design, build, and test libraries of synthetic TALE-activated promoters (STAPs) that show a broad range of expression levels in plants. In this chapter, we present protocols for the construction of artificial TALEs and corresponding STAPs.
Bücher und Buchkapitel

Marillonnet, S.; Werner, S.; Assembly of Multigene Constructs Using Golden Gate Cloning (Castilho, A., ed.). Methods Mol. Biol. 1321, 269-284, (2015) ISBN: 978-1-4939-2760-9 DOI: 10.1007/978-1-4939-2760-9_19

Efficient DNA assembly methods are required for synthetic biology. Standardization of DNA parts is an essential element that not only facilitates reuse of the same parts for various constructs but also allows standardization of the assembly strategy. We provide here a protocol for assembly of multigene constructs from standard biological parts using the modular cloning system MoClo. Making constructs using this system requires to first define the structure of the final construct and to identify all basic parts and vectors required for the construction strategy. The cloning strategy is in large part determined by the structure of the final construct, which is then made using a series of one-pot Golden Gate cloning reactions.
Bücher und Buchkapitel

Thieme, F.; Marillonnet, S.; Quick and Clean Cloning (Valla, S. & Lale, R., eds.). Methods Mol. Biol. 1116, 37-48, (2014) ISBN: 978-1-62703-764-8 DOI: 10.1007/978-1-62703-764-8_3

Identification of unknown sequences that flank known sequences of interest requires PCR amplification of DNA fragments that contain the junction between the known and unknown flanking sequences. Since amplified products often contain a mixture of specific and nonspecific products, the quick and clean (QC) cloning procedure was developed to clone specific products only. QC cloning is a ligation-independent cloning procedure that relies on the exonuclease activity of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. A specific feature of QC cloning is the use of vectors that contain a sequence called catching sequence that allows cloning specific products only. QC cloning is performed by a one-pot incubation of insert and vector in the presence of T4 DNA polymerase at room temperature for 10 min followed by direct transformation of the incubation mix in chemo-competent Escherichia coli cells.
Bücher und Buchkapitel

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.
Bücher und Buchkapitel

Balcke, G. U.; Bennewitz, S.; Zabel, S.; Tissier, A.; Isoprenoid and Metabolite Profiling of Plant Trichomes (Rodríguez-Concepción, M., ed.). Methods Mol. Biol. 1153, 189-202, (2014) ISBN: 978-1-4939-0606-2 DOI: 10.1007/978-1-4939-0606-2_13

Plant glandular trichomes are specialized secretory structures located on the surface of the aerial parts of plants with large biosynthetic capacity, often with terpenoids as output molecules. The collection of plant trichomes requires a method to separate trichomes from leaf epidermal tissues. For metabolite profiling, trichome tissue needs to be rapidly quenched in order to maintain the indigenous state of intracellular intermediates. Appropriate extraction and chromatographic separation methods must be available, which address the wide-ranging polarity of metabolites. In this chapter, a protocol for trichome harvest using a frozen paint brush is presented. A work flow for broad-range metabolite profiling using LC-MS2 analysis is described, which is applicable to assess very hydrophilic isoprenoid precursors as well as more hydrophobic metabolites from trichomes and other plant tissues.
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