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 (lambda Red)-mediated recombineering, such as the assembly of strains for biotechnological applications. Currently, the widely used lambda Red-expressing plasmids use a temperature-sensitive origin of replication or temperature shift control of lambda Red 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-beta-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(K622)(A) allele that suppresses methyl-directed mismatch repair (MMR). The coexpression of lambda Red and MutS(K622A) provides efficient oligonucleotide-mediated recombineering in the presence of active host MMR. The expression of lambda Red 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 lambda Red expression by L-rhamnose allow for all procedures to be performed at 37 degrees C. Thus, the new plasmids are robust, convenient, and versatile tools for Escherichia coli genome editing.