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. © Humana Press Inc.
CITATION STYLE
Herring, C. D. (2007). Introduction of conditional lethal amber mutations in Escherichia coli. Methods in Molecular Biology, 416, 323–334. https://doi.org/10.1007/978-1-59745-321-9_21
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