We have developed an easy, reliable two-step method for the insertion of large DNA fragments into any desired location in the E. coli chromosome. The method is based on the recombineering of a small (∼1.3 kbp) "Landing Pad" into the chromosome at the insertion site, to which the large construct is subsequently delivered via I-SceI endonuclease excision from a donor plasmid. To demonstrate the power of this method, we here show the insertion of a fragment containing the entire lac operon (∼9 kbp) into four predefined novel locations in the E. coli chromosome, a feat not possible with existing technologies. In addition, the chromosomal breaks induced by landing pad excision provide sufficient selective pressure that positive selection by antibiotics is unnecessary, making precise, exact insertion without extraneous sequence possible.
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