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Cell and Metabolic Biology
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Publications - Cell and Metabolic Biology
Publications
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
Precise annotation of time and spatial distribution of enzymes involved in plant secondary metabolism by gel electrophoresis are usually difficult due to their low abundance. Therefore, effective methods to enrich these enzymes are required to correlate available transcript and metabolite data with the actual presence of active enzymes in wild-type and mutant plants or to monitor variations of these enzymes under various types of biotic and abiotic stress conditions. S-Adenosyl-L-methionine-dependent O-methyltransferases play important roles in the modification of natural products such as phenylpropanoids or alkaloids. In plants they occur as small superfamilies with defined roles for each of its members in different organs and tissues. We explored the use of S-adenosyl-L-homocysteine as a selectivity function in affinity-based protein profiling supported by capture compound mass spectrometry. Due to their high affinity to this ligand it was possible to identify developmental changes of flower-specific patterns of plant natural product O-methyltransferases and corroborate the absence of individual O-methyltransferases in the corresponding Arabidopsis knockout lines. Developmental changes in the OMT pattern were correlated with transcript data obtained by qPCR.
Publications
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Publications
In our studies on tyrosinase-catalyzed tyrosine hydroxylation, possibly involved in betalain biosynthesis, we have evaluated different assays for the detection and quantification of the enzymatic product Dopa with respect to sensitivity, simplicity, and suitability for automatization. A tyrosinase assay including reversed-phase high-performance liquid chromatography with isocratic elution and fluorescence detection has been developed (native fluorescence of Dopa; excitation at 281 nm, emission at 314 nm). This improved assay was sensitive (detection limit: 2 pmol Dopa) and showed a wide linear range of Dopa detection (10 pmol–20 nmol Dopa). The method proved to be suitable for high-performance liquid chromatography with an autosampler and has been applied for measuring tyrosinase activity of cell cultures and different tissues ofPortulaca grandiflora.
This page was last modified on 27 Jan 2025 .
