Chromosomal recombinant gene expression offers a number of advantages over plasmid-based synthetic biology. However, the methods applied for bacterial genome engineering are still challenging and far from being standardized. Here, in an attempt to realize the simplest recombinant genome technology imaginable and facilitate the transition from recombinant plasmids to genomes, we create a simplistic methodology and a comprehensive strain collection called the Standardized Genome Architecture (SEGA). In its simplest form, SEGA enables genome engineering by combining only two reagents: a DNA fragment that can be ordered from a commercial vendor and a stock solution of bacterial cells followed by incubation on agar plates. Recombinant genomes are identified by visual inspection using green-white colony screening akin to classical blue-white screening for recombinant plasmids. The modular nature of SEGA allows precise multi-level control of transcriptional, translational, and post-translational regulation. The SEGA architecture simultaneously supports increased standardization of genetic designs and a broad application range by utilizing well-characterized parts optimized for robust performance in the context of the bacterial genome. Ultimately, its adaption and expansion by the scientific community should improve predictability and comparability of experimental outcomes across different laboratories.
Bibliographical noteFunding Information:
The authors thank Christopher Voigt (Massachusetts Institute of Technology, Cambridge, MA, USA) for providing the Marionette sensor collection upon request as well as Lars Ellgaard (Copenhagen University, Copenhagen, Denmark) and the Morten Sommer lab (Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark) for providing the plasmids pET39-Ub (His10) and pmskl2 and pZE21-RFP, respectively. Furthermore, we want to thank Cristina Hernández Rollán (Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark) for providing the plasmid pET28a-sfgfp. The authors also want to thank Joen Haahr Jensen, Jacob Søholm Mejlsted, and David Lokjær Faurdal for their support on constructing strains that contain promoters from the Marionette collection. The authors acknowledge funding by the Novo Nordisk Foundation (NNF20CC0035580) and by the Bioroboost project under EU Horizon 2020 research and innovation program under grant agreement N820699. A.K.E. was supported by grant no. NNF18CC0033664 as a fellow of the Copenhagen Bioscience PhD Programme.
© 2021, The Author(s).