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Books and chapters
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.
Books and chapters
Determination of the general capacity of proteolytic activity of a certain cell or tissue type can be crucial for an assessment of various features of an organism’s growth and development and also for the optimization of biotechnological applications. Here, we describe the use of chimeric protein stability reporters that can be detected by standard laboratory techniques such as histological staining, selection using selective media or fluorescence microscopy. Dependent on the expression of the reporters due to the promoters applied, cell- and tissue-specific questions can be addressed. Here, we concentrate on methods which can be used for large-scale screening for protein stability changes rather than for detailed protein stability studies.
Books and chapters
Ubiquitination is a central posttranslational modification that impinges on the fate of proteins. While attachment of K48-linked chains onto soluble proteins marks them for proteolysis via the 26S proteasome, mono-ubiquitination or K63-linked chains result in the endocytosis and sorting through the endomembrane system of integral membrane proteins, such as pattern recognition receptors. In vitro ubiquitination assays allow the biochemical analysis of all individual components of the ubiquitination machinery and its potential substrates. Here, we describe how to reconstitute the ubiquitination cascade in vitro and detail different variations of the assay, the required controls and how to interpret the obtained results.
Books and chapters
Conditional modulation of biological processes plays key roles in basic and applied research and in translation. It can be achieved on various levels via a multitude of approaches. One of the directions is manipulating target protein levels and activity by transcriptional, posttranscriptional, translational, and posttranslational control. Because in most of these techniques, the synthesis of the target proteins is adjusted to the needs, they all rely on the specific half-life of the target protein and its turn-over. Therefore, their time-of-action, in direct correlation to the desired reprogramming of molecular phenotypes caused by altering the target levels, is fixed and determined by the naturally inherent properties. We have introduced the low-temperature degron (lt-degron) to various intact multicellular organisms which allows to control target protein levels and therefore function and activity directly on the level of active protein. The lt-degron uses a combination of Ubiquitin-fusion technique linking target protein degradation to the N-end rule pathway of targeted proteolysis coupled with the use of cell- and tissue-specific promoters.