Synthetische Biologie

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Publikation

Kowarschik, K.; Hoehenwarter, W.; Marillonnet, S.; Trujillo, M.; UbiGate: a synthetic biology toolbox to analyse ubiquitination New Phytol. 217, 1749-1763, (2018) DOI: 10.1111/nph.14900

Ubiquitination is mediated by an enzymatic cascade that results in the modification of substrate proteins, redefining their fate. This post‐translational modification is involved in most cellular processes, yet its analysis faces manifold obstacles due to its complex and ubiquitous nature. Reconstitution of the ubiquitination cascade in bacterial systems circumvents several of these problems and was shown to faithfully recapitulate the process.Here, we present UbiGate − a synthetic biology toolbox, together with an inducible bacterial expression system – to enable the straightforward reconstitution of the ubiquitination cascades of different organisms in Escherichia coli by ‘Golden Gate’ cloning.This inclusive toolbox uses a hierarchical modular cloning system to assemble complex DNA molecules encoding the multiple genetic elements of the ubiquitination cascade in a predefined order, to generate polycistronic operons for expression.We demonstrate the efficiency of UbiGate in generating a variety of expression elements to reconstitute autoubiquitination by different E3 ligases and the modification of their substrates, as well as its usefulness for dissecting the process in a time‐ and cost‐effective manner.

Publikation

Patron, N. J.; Orzaez, D.; Marillonnet, S.; Warzecha, H.; Matthewman, C.; Youles, M.; Raitskin, O.; Leveau, A.; Farré, G.; Rogers, C.; Smith, A.; Hibberd, J.; Webb, A. A. R.; Locke, J.; Schornack, S.; Ajioka, J.; Baulcombe, D. C.; Zipfel, C.; Kamoun, S.; Jones, J. D. G.; Kuhn, H.; Robatzek, S.; Van Esse, H. P.; Sanders, D.; Oldroyd, G.; Martin, C.; Field, R.; O'Connor, S.; Fox, S.; Wulff, B.; Miller, B.; Breakspear, A.; Radhakrishnan, G.; Delaux, P.-M.; Loqué, D.; Granell, A.; Tissier, A.; Shih, P.; Brutnell, T. P.; Quick, W. P.; Rischer, H.; Fraser, P. D.; Aharoni, A.; Raines, C.; South, P. F.; Ané, J.-M.; Hamberger, B. R.; Langdale, J.; Stougaard, J.; Bouwmeester, H.; Udvardi, M.; Murray, J. A. H.; Ntoukakis, V.; Schäfer, P.; Denby, K.; Edwards, K. J.; Osbourn, A.; Haseloff, J.; Standards for plant synthetic biology: a common syntax for exchange of DNA parts New Phytol. 208, 13-19, (2015) DOI: 10.1111/nph.13532

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease‐mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.