Integration-helper plasmid mediated rapid gene knock-out in E. coli

Abstract

In this work, an integration helper plasmid was designed for rapid gene manipulation inE. coli. This plasmid (pCW611) expresses two recombinases (Red and Cre) underseparate inducible promoters (PlacUV5 and PBAD). Thus, iterative plasmid transformationand curing is avoided, resulting in significant time savings compared to the traditional,two-plasmid approach. This enabled target gene deletion in 3 days. The system wasverified by deleting adhE, sfcA, frdABCD, and ackA individualy and pairwise (adhEaspAand sfcA-aspA). Finally, a fumaric acid producing E. coil strain was developed bydeleting four genes (fumB, iclR, fumA, and fumC) in 10 days as a proof-of-concept.[This work was supported by the Technology Development Program to Solve ClimateChanges on Systems Metabolic Engineering for Biorefineries from the Ministry ofScience, ICT and Future Planning (MSIP) through the National Research Foundation(NRF) of Korea. MG was additionallysupported by the Swedish research council Formas].