Development of new versatile plasmid-based systems for λRed-mediated Escherichia coli genome engineering

Plasmid-based systems are the most appropriate for multistep lambda Red (λRed)-mediated recombineering, such as the assembly of strains for biotechnological applications. Currently, the widely used λRed-expressing plasmids use a temperature-sensitive origin of replication or temperature shift control of λRed expression. In this work, we have constructed a new, conditionally replicating vector that can be efficiently eliminated from the host strain through passaging in medium containing isopropyl-β-d-thiogalactopyranoside. Using the new vector, we have developed two improved helper plasmids (viz., pDL17 and pDL14) for dsDNA and oligonucleotide-mediated recombineering, respectively. The plasmid pDL14 contains a dominant negative mutS^K622A allele that suppresses methyl-directed mismatch repair (MMR). The coexpression of λRed and mutS^K622A provides efficient oligonucleotide-mediated recombineering in the presence of active host MMR. The expression of λRed was placed under the control of the tightly regulated P_rhaB promoter. Because of their low expression level under uninduced conditions, both plasmids could be maintained without elimination for multiple recombineering steps. The temperature-independent replication of the plasmids and control of λRed expression by l-rhamnose allow for all procedures to be performed at 37 °C. Thus, the new plasmids are robust, convenient, and versatile tools for Escherichia coli genome editing.