Efficient Bacterial Genome Engineering throughout the Central Dogma Using the Dual-Selection Marker tetAOPT

Carolyn N. Bayer, Ana G.V. Sepulchro, Maja Rennig, Morten H.H. Nørholm*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

Engineering of bacterial genomes is a fundamental craft in contemporary biotechnology. The ability to precisely edit chromosomes allows for the development of cells with specific phenotypes for metabolic engineering and for the creation of minimized genomes. Genetic tools are needed to select for cells that underwent editing, and dual-selection markers that enable both positive and negative selection are highly useful. Here, we present an optimized and easy-to-use version of the tetA dual-selection marker and demonstrate how this tetAOPTcan be used efficiently to engineer at different stages of the central dogma of molecular biology. On the DNA level, tetAOPTcan be used to create scarless knockouts across the Escherichia coli genome with efficiency above 90%, whereas recombinant gene integrations can be achieved with approximately 50% efficiency. On the RNA and protein level, we show that tetAOPTenables advanced genome engineering of both gene translation and transcription by introducing sequence variation in the translation initiation region or by exchanging promoters. Finally, we demonstrate the use of tetAOPTfor genome engineering in the industrially relevant probiotic strain E. coli Nissle.

Original languageEnglish
JournalACS Synthetic Biology
Volume11
Issue number10
Pages (from-to)3440-3450
ISSN2161-5063
DOIs
Publication statusPublished - 2022

Keywords

  • Dual-selection marker
  • Escherichia coli
  • Escherichia coli Nissle
  • Genome engineering
  • Recombineering
  • tetA

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