Protein engineers and synthetic biologists generally rely upon Escherichia coli, a choice that leads to a life of cloning. Acinetobacter baylyi sp. ADP1 is distantly related to E. coli, but offers two significant advantages over its more famous cousin. A. baylyi is naturally competent; all cells in a growing population can import double-stranded DNA, including PCR products, from their growth media. A. baylyi recombines DNA very efficiently, so sequences similar to those in the chromosome are efficiently integrated. Here we show how these natural advantages obviate cloning and streamline the directed evolution of genes and genomes. A native promoter (pobA) and reporter gene (the E. coli gusA) were integrated into the Acinetobacter chromosome. The resulting strain was evolved in six rounds of serial passage (without PCR or cloning) and screening for clones with increased beta-glucuronidase (GUS) activity. Quantitative PCR analysis showed that the evolved chromosome contained ~240 copies of the promoter-reporter cassette (collectively over half the size to the original chromosome). In a separate experiment, an E. coli-A. baylyi shuttle plasmid encoding the same promoter and reporter was evolved in two rounds of Rolling Circle Amplification (again, without PCR or cloning) and high throughput screening. A. baylyi thus enables facile directed evolution algorithms that would not be possible with E. coli.

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