Simple and robust in vivo engineering of plasmid DNA at any copy number in Escherichia coli

Plasmids and the model bacterium Escherichia coli are at the heart of recombinant gene technologies. Plasmids are handled in test tubes with enzymes such as restriction endonucleases, ligases, and polymerases. However, with the increasing demand for larger and more complex designs, in vitro manipulation constitutes a bottleneck. By combining recombination with genetic selection, in vivo manipulation of genomic DNA is becoming routine but is yet to be developed as a versatile and reliable way to make plasmid DNA. Here, we present a robust methodology for plasmid recombineering in E. coli using a triple-selection system customized for efficient performance at any copy number. Equipped with this genetic selection cassette, we generate a toolbox of plasmids in a standardized framework with popular genetic modules. By reducing the time and resources for making recombinant DNA, this approach should enable automation and accelerate the development of biological solutions.