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.
Herring
Christopher D
CD
Mascoma Corporation, Lebanon, NH, USA.
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D020043:Genes, Essential; D016153:Genes, Suppressor; D009154:Mutation; D012329:RNA, Bacterial / Q000235:genetics*; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics*
D000900:Anti-Bacterial Agents; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl