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Suberin is a hydrophobic biopolymer that acts as an internal and external diffusion and transpiration barrier in plants. It is involved in two phases of wound healing, i.e. initial closing layer formation and subsequent wound periderm development. Transcriptomic and metabolomic analyses of wounded potato leaf tissue revealed preferential induction of cell wall modifying processes during closing layer formation, accompanied by a highly active defense response. To address the importance of suberin in this process, we generated loss of function mutants by CRISPR-Cas9 editing the suberin transporter gene StABCG1. Both wound-induced StABCG1 transcript levels and suberin formation around wounded leaf tissue were reduced in CRISPR-lines. Moreover, wound-induced tissue damage was characterized by browning of wound-adjacent areas. Transcriptome analyses of these areas revealed up-regulation of genes encoding defense proteins and enzymes of the phenylpropanoid pathway. Levels of hydroxycinnamic acid amides, acting in defense and in cell wall reinforcement, were drastically enhanced in CRISPR compared to control plants. These results suggest that the reduction in suberin formation around wounded tissue leads to a loss of barrier function, resulting in tissue browning due to enhanced exposure to oxygen.
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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.
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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.