Abstract
Escherichia coli K-12 is one of the most well-studied species of bacteria. This species, however, is much more difficult to modify by homologous recombination (HR) than other model microorganisms. Research on HR in E. coli has led to a better understanding of the molecular mechanisms of HR, resulting in technical improvements and rapid progress in genome research, and allowing whole-genome mutagenesis and large-scale genome modifications. Developments using λ Red (exo, bet, and gam) and CRISPR-Cas have made E. coli as amenable to genome modification as other model microorganisms, such as Saccharomyces cerevisiae and Bacillus subtilis. This review describes the history of recombination research in E. coli, as well as improvements in techniques for genome modification by HR. This review also describes the results of large-scale genome modification of E. coli using these technologies, including DNA synthesis and assembly. In addition, this article reviews recent advances in genome modification, considers future directions, and describes problems associated with the creation of cells by design.