期刊
GENE
卷 311, 期 -, 页码 153-163出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/S0378-1119(03)00585-7
关键词
lambda red recombination; mutagenesis; homing endonuclease; I-Sce I; suppressor tRNA; markerless
资金
- NIGMS NIH HHS [5T32 GM 08349, GM 35682] Funding Source: Medline
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. (C) 2003 Elsevier Science B.V. All rights reserved.
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