In the fast lane: Large-scale bacterial genome engineering

The last few years have witnessed rapid progress in bacterial genome engineering. The long-established, standard ways of DNA synthesis, modification, transfer into living cells, and incorporation into genomes have given way to more effective, large-scale, robust genome modification protocols. Expansion of these engineering capabilities is due to several factors. Key advances include: (i) progress in oligonucleotide synthesis and in vitro and in vivo assembly methods, (ii) optimization of recombineering techniques, (iii) introduction of parallel, large-scale, combinatorial, and automated genome modification procedures, and (iv) rapid identification of the modifications by barcode-based analysis and sequencing. Combination of the brute force of these techniques with sophisticated bioinformatic design and modeling opens up new avenues for the analysis of gene functions and cellular network interactions, but also in engineering more effective producer strains. This review presents a summary of recent technological advances in bacterial genome engineering.

► We review recent results in high throughput genome engineering. ► Large scale synthesis of oligonucleotides, and assembly into genes and genomes. ► Highly parallel barcoded modifications of genomes. ► Combinatorial modification of bacterial genomes by recombineering. ► Merging of modified genomic segments by conjugation.