Unser 10. Leibniz Plant Biochemistry Symposium am 7. und 8. Mai war ein großer Erfolg. Thematisch ging es in diesem Jahr um neue Methoden und Forschungsansätze der Naturstoffchemie. Die exzellenten Vorträge über Wirkstoffe…
Omanische Heilpflanze im Fokus der Phytochemie IPB-Wissenschaftler und Partner aus Dhofar haben jüngst die omanische Heilpflanze Terminalia dhofarica unter die phytochemische Lupe genommen. Die Pflanze ist reich an…
Geschmack ist vorhersagbar: Mit FlavorMiner. FlavorMiner heißt das Tool, das IPB-Chemiker und Partner aus Kolumbien jüngst entwickelt haben. Das Programm kann, basierend auf maschinellem Lernen (KI), anhand der…
Marillonnet, S.; Werner, S.;Golden gate cloning of multigene constructs using the modular cloning system MoCloSchindler D.Methods Mol. Biol.285021-39(2025)ISBN:978-1-0716-4094-4DOI: 10.1007/978-1-0716-4220-7_2
Modular cloning systems that rely on type IIS enzymes for DNA assembly have many advantages for construct engineering for biological research and synthetic biology. 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 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
Grützner, R.; Marillonnet, S.;Golden gate cloning of MoClo standard partsSchindler D.Methods Mol. Biol.28501-19(2025)ISBN:978-1-0716-4094-4DOI: 10.1007/978-1-0716-4220-7_1
Efficient DNA assembly methods are an essential prerequisite in the field of synthetic biology. Modular cloning systems, which rely on Golden Gate cloning for DNA assembly, are designed to facilitate assembly of multigene constructs from libraries of standard parts through a series of streamlined one-pot assembly reactions. Standard parts consist of the DNA sequence of a genetic element of interest such as a promoter, coding sequence, or terminator, cloned in a plasmid vector. Standard parts for the modular cloning system MoClo, also called level 0 modules, must be flanked by two BsaI restriction sites in opposite orientations 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 level 0 modules. This protocol requires the following steps: (1) defining the type of part that needs to be cloned, (2) designing primers for amplification, (3) performing polymerase chain reaction (PCR) amplification, (4) cloning of the fragments using Golden Gate cloning, and finally (5) sequencing of the part. For large standard parts, it is preferable to first clone sub-parts as intermediate level-1 constructs. These sub-parts are sequenced individually and are then further assembled to make the final level 0 module.
Publikation
Heuermann, D.; Döll, S.; Schweneker, D.; Feuerstein, U.; Gentsch, N.; von Wirén, N.;Distinct metabolite classes in root exudates are indicative for field- or hydroponically-grown cover cropsFront. Plant Sci.141122285(2023)DOI: 10.3389/fpls.2023.1122285
Introduction: Plants release a large variety of metabolites via their roots to shape physico-chemical soil properties and biological processes in the rhizosphere. While hydroponic growth conditions facilitate accessibility of the root system and recovery of root exudates, the natural soil environment can alter root metabolism and exudate secretion, raising the question to what extent the quantity and composition of root exudates released in hydroponic growth systems reflect those recovered from soil-grown roots.
Methods: Using a root washing method, we sampled root exudates from four field-grown cover crop species with wide taxonomic distance, namely white mustard, lacy phacelia, bristle oat, and Egyptian clover. A set of primary metabolites and secondary metabolites were analysed in a targeted and untargeted LC-MS-based approach, respectively, for comparison with exudates obtained from hydroponically cultured plants.
Results and discussion: We found that hydroponically cultivated plants released a larger amount of total carbon, but that the recovery of total carbon was not indicative for the diversity of metabolites in root exudates. In the field, root exudates from phacelia and clover contained 2.4 to 3.8 times more secondary metabolites, whereas carbon exudation in hydroponics was 5- to 4-fold higher. The composition of the set of metabolites identified using the untargeted approach was much more distinct among all species and growth conditions than that of quantified primary metabolites. Among secondary metabolite classes, the presence of lipids and lipid-like molecules was highly indicative for field samples, while the release of a large amount of phenylpropanoids, organoheterocyclic compounds or benzenoids was characteristic for clover, mustard or oat, respectively, irrespective of the cultivation condition. However, at the compound level the bulk of released metabolites was specific for cultivation conditions in every species, which implies that hydroponically sampled root exudates poorly reflect the metabolic complexity of root exudates recovered from field-grown plants.
Publikation
Paponov, M.; Ziegler, J.; Paponov, I. A.;Light exposure of roots in aeroponics enhances the accumulation of phytochemicals in aboveground parts of the medicinal plants Artemisia annua and Hypericum perforatumFront. Plant Sci.141079656(2023)DOI: 10.3389/fpls.2023.1079656
Light acts as a trigger to enhance the accumulation of secondary compounds in the aboveground part of plants; however, whether a similar triggering effect occurs in roots is unclear. Using an aeroponic setup, we investigated the effect of long-term exposure of roots to LED lighting of different wavelengths on the growth and phytochemical composition of two high-value medicinal plants, Artemisia annua and Hypericum perforatum. In A. annua, root exposure to white, blue, and red light enhanced the accumulation of artemisinin in the shoots by 2.3-, 2.5-, and 1.9-fold, respectively. In H. perforatum, root exposure to white, blue, red, and green light enhanced the accumulation of coumaroylquinic acid in leaves by 89, 65, 84, and 74%, respectively. Root lighting also increased flavonol concentrations. In contrast to its effects in the shoots, root illumination did not change phytochemical composition in the roots or root exudates. Thus, root illumination induces a systemic response, resulting in modulation of the phytochemical composition in distal tissues remote from the light exposure site.