An electroporation-free method based on Red recombineering for markerless deletion and genomic replacement in the Escherichia coli DH1 genome.

ABSTRACT: The λ-Red recombination system is a popular method for gene editing. However, its applications are limited due to restricted electroporation of DNA fragments. Here, we present an electroporation-free λ-Red recombination method in which target DNA fragments are excised by I-CreI endonuclease in vivo from the landing pad plasmid. Subsequently, the I-SceI endonuclease-cutting chromosome and DNA double-strand break repair were required. Markerless deletion and genomic replacement were successfully accomplished by this novel approach. Eight nonessential regions of 2.4-104.4 kb in the Escherichia coli DH1 genome were deleted separately with selection efficiencies of 5.3-100%. Additionally, the recombination efficiencies were 2.5-45%, representing an order of magnitude improvement over the electr

SUBMITTER: Wei Y 

PROVIDER: S-EPMC5655456 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature