title: SEVA Linkers: A Versatile and Automatable DNA Backbone Exchange Standard for Synthetic Biology.
abstract: 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.
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Hørsholm DK-2970, Denmark.
authors.forename: Se Hyeuk
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Hørsholm DK-2970, Denmark.
authors.forename: Ana Mafalda
authors.initials: AM
authors.lastname: Cavaleiro
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Hørsholm DK-2970, Denmark.
authors.forename: Maja
authors.initials: M
authors.lastname: Rennig
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Hørsholm DK-2970, Denmark.
authors.forename: Morten H H
authors.initials: MH
authors.lastname: Nørholm
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D053938:DNA Fragmentation; D004261:DNA Replication / Q000187:drug effects; D004269:DNA, Bacterial / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D005818:Genetic Engineering; D005822:Genetic Vectors; D007763:Lac Operon; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D058615:Synthetic Biology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
keywords: cell factory design; plasmid backbone exchange; standard parts characterization; synthetic biology standards