Application of Cloning-Free Genome Engineering to Escherichia coli .

The propagation of foreign DNA in is central to molecular biology. Recent advances have dramatically expanded the ability to engineer (bacterial) cells; however, most of these techniques remain time-consuming. The aim of the present work was to explore the possibility to use the cloning-free genome editing (CFGE) approach, proposed by Döhlemann and coworkers (2016), for genetics, and to deepen the knowledge about the homologous recombination mechanism. The auxotrophic mutant strains FB182 () and FB181 () were transformed with the circularized wild-type (i) gene and gene fragments of decreasing length, and (ii) gene, respectively. His clones were selected based on their ability to grow in the absence of histidine, and their / gene sequences were characterized. CFGE method allowed the recombination of wild-type genes (or fragments of them) within the mutated chromosomal copy, with a different recombination frequency based on the fragment length, and the generation of clones with a variable number of in tandem genes copies. Data obtained pave the way to further evolutionary studies concerning the homologous recombination mechanism and the fate of in tandem duplicated genes.