Work

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

Year: 2025

Type: article

Abstract: 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. A robust in vivo method for engineering plasmid DNA in E. coli using a triple-selection system that works at any copy number, enabling fast, scalable construction of standardized plasmids for synthetic biology applications.

Source: Communications Biology

DOI: 10.1038/s42003-025-08361-9

Authors: Ana G. V. Sepulchro, Hendrikje C. J. Kozlowski, Morten H. H. Nørholm

Institutions: Novo Nordisk Foundation, Technical University of Denmark

Language: English

Topic: Microbial Metabolic Engineering and Bioproduction

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $4,190

Funders: Novo Nordisk, Stockholms Universitet, Novo Nordisk Fonden