PMID:12853150

Citation	Herring, CD, Glasner, JD and Blattner, FR (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311:153-63
Abstract	We have developed a method called "gene gorging" to make precise mutations in the Escherichia coli genome at frequencies high enough (1-15%) to allow direct identification of mutants by PCR or other screen rather than by selection. Gene gorging begins by establishing a donor plasmid carrying the desired mutation in the target cell. This plasmid is linearized by in vivo expression of the meganuclease I-SceI, providing a DNA substrate for lambda Red mediated recombination. This results in efficient replacement of the wild type allele on the chromosome with the modified sequence. We demonstrate gene gorging by introducing amber stop codons into the genes xylA, melA, galK, fucI, citA, ybdO, and lacZ. To compliment this approach we developed an arabinose inducible amber suppressor tRNA. Controlled expression mediated by the suppressor was demonstrated for the lacZ and xylA amber mutants.
Links	PubMed
Keywords	Anti-Bacterial Agents/pharmacology; Chloramphenicol/pharmacology; Codon, Terminator/genetics; Drug Resistance, Bacterial; Escherichia coli/drug effects; Escherichia coli/genetics; Escherichia coli/growth & development; Gene Expression Regulation, Bacterial; Genes, Bacterial/genetics; Genes, Suppressor; Genome, Bacterial; Kanamycin/pharmacology; Mutagenesis, Insertional/methods; Mutation; Plasmids/genetics; Recombination, Genetic/genetics; Selection, Genetic; Suppression, Genetic/genetics
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