Introduction of Conditional Lethal Amber Mutations in Escherichia coli
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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-Sce I 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.
