Natural transformation as a tool in Acinetobacter baylyi: Evolution by amplification of gene copy number

For many years, the natural competency of Acinetobacter baylyi ADP1 facilitated studies of bacterial metabolism, biochemistry, and physiology. With the advent of synthetic biology, new opportunities arise to exploit the remarkable transformability and chromosomal plasticity of this model organism. In this chapter, we describe a recently developed method, “Evolution by Amplification and Synthetic Biology” (EASy). EASy allows the targeted amplification of chromosomal segments that give rise to new phenotypes. Increased gene dosage regulates protein expression in a rudimentary fashion by establishing a chromosomal array in which copy number adjusts via recombination between repeated DNA sequences. Selective conditions enrich for cells within the population that confer a growth advantage. Under continuous selective pressure, beneficial mutations may accumulate in any genomic region. Such mutations favour decreases in the average copy number of the target region. Thus, the genetic flexibility afforded by transient copy number variation helps accelerate the selection of engineered strains with desired traits during laboratory evolution. Thanks to the extremely simple genetic manipulation of A. baylyi ADP1, the EASy method can be readily implemented by researchers without the need for advanced instrumentation or complex cloning techniques.