Abstract  Strain engineering, like cloning, is a fundamental technology used to confer new traits onto existing strains. While effective methods for trait development through gene modification within strains have been developed, methods for trait transfer between Escherichia coli strains to create complex strains are needed. We report herein the development of genome mass transfer (GMT), a broadly applicable new strain engineering methodology enabling rapid trait transfer from a donor strain into a recombineering gene-expressing recipient strain. GMT utilizes electroporation of donor chromosomal DNA into a recombineering recipient cell for precise trait transfer. GMT transfer of traits between E. coli strains can be used to rapidly assemble new strains incorporating combinations of marked gene knockouts, for example, utilizing the existing E. coli K-12 Keio gene knockout collection as source target genes. Optional use of random primed isothermal amplified DNA eliminates the need for initial DNA purification, affording high throughput application. This allows unprecedented simplicity and speed for rational design engineering of complex phenotypes in industrial strains.