Kim Se Hyeuk, Cavaleiro Ana Mafalda, Rennig Maja, Nørholm Morten H H
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Hørsholm DK-2970, Denmark.
ACS Synth Biol. 2016 Oct 21;5(10):1177-1181. doi: 10.1021/acssynbio.5b00257. Epub 2016 Mar 4.
DNA vectors serve to maintain and select recombinant DNA in cell factories, and as design complexity increases, there is a greater need for well-characterized parts and methods for their assembly. Standards in synthetic biology are top priority, but standardizing molecular cloning contrasts flexibility, and different researchers prefer and master different molecular technologies. Here, we describe a new, highly versatile and automatable standard "SEVA linkers" for vector exchange. SEVA linkers enable backbone swapping with 20 combinations of classical enzymatic restriction/ligation, Gibson isothermal assembly, uracil excision cloning, and a nicking enzyme-based methodology we term SEVA cloning. SEVA cloning is a simplistic one-tube protocol for backbone swapping directly from plasmid stock solutions. We demonstrate the different performance of 30 plasmid backbones for small molecule and protein production and obtain more than 10-fold improvement from a four-gene biosynthetic pathway and 430-fold improvement with a difficult-to-express membrane protein. The standardized linkers and protocols add to the Standard European Vectors Architecture (SEVA) resource and are freely available to the synthetic biology community.
DNA载体用于在细胞工厂中维持和筛选重组DNA，随着设计复杂性的增加，对特征明确的组件及其组装方法的需求也越来越大。合成生物学中的标准是首要任务，但分子克隆的标准化与灵活性相悖，不同的研究人员偏爱并掌握不同的分子技术。在此，我们描述了一种用于载体交换的全新、高度通用且可自动化的标准“SEVA接头”。SEVA接头能够通过20种经典酶切/连接、吉布森等温组装、尿嘧啶切除克隆以及一种我们称为SEVA克隆的基于切口酶的方法进行骨架交换。SEVA克隆是一种简单的单管方案，可直接从质粒储备溶液中进行骨架交换。我们展示了30种质粒骨架在小分子和蛋白质生产方面的不同性能，并且从一个四基因生物合成途径中获得了超过10倍的改进，对于一种难以表达的膜蛋白则有430倍的改进。这些标准化的接头和方案丰富了标准欧洲载体架构（SEVA）资源，可供合成生物学界免费使用。
