Efficient Bacterial Genome Engineering throughout the Central Dogma Using the Dual-Selection Marker tetA(OPT)

[Image: see text] 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 tetA(OPT) can be used efficiently to engineer at different stages of the central dogma of molecular biology. On the DNA level, tetA(OPT) can 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 tetA(OPT) enables 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 tetA(OPT) for genome engineering in the industrially relevant probiotic strain E. coli Nissle.