Genomic replacement in Escherichia coli K-12 using covalently closed circular plasmid DNA

A number of gene replacements at different loci were constructed using covalently closed circular (ccc) plasmid DNA in he recB21 recC22 sbcB15 asbcC201 mutant of Escherichia coli (JC7623). Selected constructs representing deletions and insertion mutations formed from double-crossover events involving the ccc plamid molecules and the genome were confirmed by Southern blots, and the frequency of double-crossover events was evaluated. It is reported that such mutants may be constructed without linearizing plasmid DNA, as described previously.