UbiGate a synthetic biology toolbox to analyse ubiquitination

UbiGate : a synthetic biology toolbox to analyse ubiquitination

© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Bibliographische Detailangaben
Veröffentlicht in:The New phytologist. - 1979. - 217(2018), 4 vom: 15. März, Seite 1749-1763
1. Verfasser: Kowarschik, Kathrin (VerfasserIn)
Weitere Verfasser: Hoehenwarter, Wolfgang, Marillonnet, Sylvestre, Trujillo, Marco
Format: Online-Aufsatz
Sprache:English
Veröffentlicht: 2018
Zugriff auf das übergeordnete Werk:The New phytologist
Schlagworte:Journal Article Research Support, Non-U.S. Gov't Golden Gate cloning bacterial expression post-translational modification synthetic biology ubiquitination Ubiquitin Ubiquitinated Proteins
Beschreibung
Zusammenfassung:© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.
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
Beschreibung:Date Completed 13.09.2019
Date Revised 30.09.2020
published: Print-Electronic
GENBANK: AY074795.1, NM_001320229
Citation Status MEDLINE
ISSN:1469-8137
DOI:10.1111/nph.14900