Designing the selection cassette for the continuous, parallel, and iterative operation of genome engineeringView all posters
Department of Applied Chemistry and Biotechnology, Japan
For the direct editing of bacterial genome with single-base resolution, two-step recombination methods are widely used. Here, the target site is replaced with PCR-generated DNA cassette first, and the cassette is then replaced with exogenous genes. The DNA cassettes code for positive/negative markers in order to select both for the intermediate/final states. Here, we report the systematic effort to re-design the system to improve the robustness, throughput, and speed of this method to realize the seamless and automation-liable platform for genome engineering. In search for the robust negative genetic selection that can be operated by liquid handling, we found the nuclease kinase activity for non-natural nucleotide dP, originally developed for the directed evolution of genetic switches/circuits (Tashiro et al., Nuc. Acids Res., 39, e12 (2010)), provides robust and efficient selection for the recombinant cell only with liquid handling. Creating/testing the chimeric selectors, duplication of selector genes, tuning of the expression level of individual selector genes enabled us to eliminate the emergence of false-positive clones during the process. Having established the repeatable workflow where all the steps can be rapidly and seamlessly operated by liquid handling, we demonstrate the multi-step and in-parallel operation of genome modifications to generates a diverse set of isoprenoid-overproducing Escherichia coli strains.