UbiGate: a synthetic biology toolbox to analyse ubiquitination

Corresponding author: Marco Trujillo

• Ubiquitination (泛素化) is mediated by an enzymatic cascade (酶促级联) that results in the modification of substrate proteins, redefining their fate. This post-translational modification (翻译后修饰) is involved in most cellular processes, yet its analysis faces manifold (各种各样的) obstacles (障碍) due to its complex and ubiquitous nature. Reconstitution (重建) 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 (合成生物学) toolbox, together with an inducible (可诱导的) bacterial expression system – to enable the straightforward reconstitution of the ubiquitination cascades of different organisms in Escherichia coli (大肠杆菌) by ‘Golden Gate’ cloning.

• This inclusive toolbox uses a hierarchical (分层的) modular cloning system to assemble complex DNA molecules encoding the multiple genetic elements of the ubiquitination cascade in a predefined (预定义的) order, to generate polycistronic operons (多顺反子) for expression.

• We demonstrate the efficiency of UbiGate in generating a variety of expression elements to reconstitute autoubiquitination by different E3 ligases (连接酶) and the modification of their substrates, as well as its usefulness for dissecting the process in a time- and cost-effective manner.

本文开发了一套分层模块化克隆系统用以组装编码泛素化级联多个遗传元件的复杂DNA分子，叫做UbiGate。各种载体信息可见https://www.addgene.org/

通讯：Marco Trujillo (http://www.trujillolab.com/team.htm)

个人简介：硕士，研究活性氧物质在谷物和白粉病互作中的作用；之后在德国Gießen University读博士依然继续硕士的研究；之后到英国Ken Shires实验室，开始转向拟南芥中泛素连接酶在免疫响应中的功能；之后，跟着Ken到了日本的RIKEN继续连接酶方面的研究；再之后Würzburg University短暂的停留后，开始创建自己的团队，并到了Halle的IPB成为了独立的青年团队领袖。

doi: 10.1111/nph.14900