Markerless gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome

A simple and efficient gene replacement method, based on the recombination and repair activities of the cell, was developed. The method permits the ta rgeted construction of markerless deletions, insertions and point mutations in the Escherichia coli chromosome. A suicide plasmid, carrying the mutant allele and the recognition site of meganuclease I-Scel, is inserted into t he genome by homologous recombination between the mutant and the wild-type (wt) alleles, Resolution of this cointegrate by intramolecular recombinatio n of the allele pair results in either a mutant or a wt chromosome which ca n be distinguished by allele-specific PCR screening. The resolution process is stimulated by introducing a unique double-strand break (DSB) into the c hromosome at the I-Scel site. Cleavage by the nuclease not only enhances th e frequency of resolution by two to three orders of magnitude, but also sel ects for the resolved products. The DSB-stimulated gene replacement method can be used in recombination-proficient E.eoli cells, does not require spec ific growth conditions, and is potentially applicable in other microorganis ms. se of the method was demonstrated by constructing a 17-bp and a 62-kb d eletion in the MIG1655 chromosome. Cleavage of the chromosome induces the S OS response but does not lead to an increased mutation rate.