Introduction of Conditional Lethal Amber Mutations in Escherichia coli

A method is described for generating conditional lethal mutations in essential genes in Escherichia coli. In this procedure, amber stop codons are introduced as “tagalong” mutations in the flanking DNA of a downstream antibiotic-resistance marker by lambda Red recombination. The marker is removed by expression of I-SceI homing endonuclease, leaving a markerless mutation. The mutants then depend upon expression of a suppressor transfer RNA (tRNA) for survival, which is expressed under control of the arabinose promoter on a high-copy-number plasmid.