Journal article
SEVA Linkers: A Versatile and Automatable DNA Backbone Exchange Standard for Synthetic Biology
Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark1
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark2
Membrane Synthetic Biology Group, Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark3
iLoop, Translational Management, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark4
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.
Language: | English |
---|---|
Year: | 2016 |
Pages: | 1177-1181 |
ISSN: | 21615063 |
Types: | Journal article |
DOI: | 10.1021/acssynbio.5b00257 |
ORCIDs: | Kim, Se Hyeuk , Rennig, Maja and Nørholm, Morten |
Cell factory design Plasmid backbone exchange Standard parts characterization Synthetic biology standards
Anti-Bacterial Agents Base Sequence Cloning, Molecular DNA Fragmentation DNA Replication DNA, Bacterial DNA-Directed RNA Polymerases Drug Resistance, Bacterial Escherichia coli Genetic Engineering Genetic Vectors Lac Operon Plasmids Promoter Regions, Genetic Protein Conformation Synthetic Biology Viral Proteins bacteriophage T7 RNA polymerase cell factory design plasmid backbone exchange standard parts characterization synthetic biology standards