Abstract
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.
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© 2008 Humana Press Inc., a part of Springer Science+Business Media, LLC
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Herring, C.D. (2008). Introduction of Conditional Lethal Amber Mutations in Escherichia coli . In: Osterman, A.L., Gerdes, S.Y. (eds) Microbial Gene Essentiality: Protocols and Bioinformatics. Methods in Molecular Biology™, vol 416. Humana Press. https://doi.org/10.1007/978-1-59745-321-9_21
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DOI: https://doi.org/10.1007/978-1-59745-321-9_21
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