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Engineering of bacterial genomes is a fundamental craft in contemporary biotechnology. The ability to precisely edit bacterial chromosomes allows for the development of cells with specific phenotypes for metabolic engineering and for 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 thetetAdual selection marker and demonstrate how this tetAOPT can be used efficiently to engineer at different stages of the central dogma of molecular biology. On the DNA level, tetAOPT can be used to create scarless knockouts across the E. coli genome with an efficiency above 90% whereas recombinant gene integrations can be achieved with approximately 50% efficiency. On the expression level, we show that tetAOPT enables advanced genome engineering of both genetranslation and transcription. Finally, we demonstrate the use of tetAOPT for genome engineering in the industrially relevant probiotic strain E. coli Nissle.
