Scarless Chromosomal Gene Knockout Methods

An improved and rapid genomic engineering method has been developed for the construction of �custom-designed microorganisms by scarless chromosomal gene knockouts. This method, which can be performed in 2 days, permits restructuring of the Escherichia coli genome via scarless deletion of selected genomic regions. The deletion process is mediated by a special plasmid, pREDI, which carries two independent inducible promoters: (1) an arabinose-inducible promoter that drives expression of λ-RED recombination proteins, which carry out the replacement of a target genomic region with a marker-containing linear DNA cassette, and (2) a rhamnose-inducible promoter that drives expression of I-Sce I endonuclease, which accomplishes deletion of the introduced marker by double-strand breakage – mediated intramolecular recombination. This genomic deletion is performed simply by changing the carbon source in the bacterial growth medium from arabinose to rhamnose. The efficiencies of targeted region replacement and deletion of the inserted linear DNA cassette are nearly 70 and 100%, respectively. This rapid and efficient procedure can be adapted for use in generating a variety of genome modifications.