Oligo- and dsDNA-Mediated Genome Editing Using a Teta Dual Selection System and Its Application in Escherichia coli

Escherichia coli has been used widely as a cell factory, which can make various value-added materials by engineering the genome or/and introducing the heterologous genes related to the target material-producing biosynthetic pathway of interests. Thus, precise and seamless genome modification method to engineer targeted genome is needed to construct potent biosynthetic system. Herein, we report a promising method in E. coli that relies on the insertion of an optimized tetA dual selection cassette followed by replacement of the same cassette with short, single-stranded DNA (oligos) or long, double−stranded DNA and isolation of recombinants by the optimized selection process on NiCl₂. This method could be rapidly and successfully used for genome engineering, including deletions, insertions, replacements, and point mutations.

By using our method, we tested the strength of various promoters by expressing gfp (Green Fluorescent Protein) under them on the chromosome using the TetA dual selection system. Previously, metabolic engineering and synthetic biology in precedent researches usually introduce target genes on plasmid due to its ability to control expression level according to their various copy number from low to high and easy introduction to desired strains. There are some limitations about its maintenance such as instability and robustness. The copy number variation of plasmid can affect consistent expression of the genes related to specific metabolic pathway, which can lead to variation of yield of target materials among the same strains. Besides, the plasmid is easy to insert into the desired strain by transformation, but also easy to escape from the strain according to condition like the origin of replication, at the same time. Thus, we have tried to combine the genome editing method and promoter engineering for constant and high level gene expression system on genome. We tested various inducible promoters, constitutive promoters and generated new promoters for genome-level gene expression. Moreover, increment and optimization of gene expression and construction of biosynthetic system on genome aim to overcome the disadvantages of plasmid-based metabolic engineering and promise stable expression on genome. In conclusion, this method can be applied to achieve scarless, proficient, and targeted genome editing for strain construction, metabolic engineering, and synthetic biology.