<i>In vivo</i> cloning of large chromosomal segments into a BAC derivative by generalized transduction and recombineering in <i>Salmonella enterica</i>
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- Kato Akinori
- Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University
説明
<p>Recombineering has been used to facilitate the development of in vivo cloning methods. However, the method relies heavily on PCR, which still generates a much higher error rate than DNA replication in vivo, even when amplifying large DNA inserts. Here, a precise technique is reported in Salmonella enterica that enables the cloning of up to at least 19 kb target chromosomal DNA segments that had been marked by FRTs, which were derived from two consecutive lambda Red-mediated recombination events. P22 phage was utilized to transduce the target DNA segments from donor strains to recipient strains harboring a derivative of bacterial artificial chromosome (BAC) containing a FRT and a plasmid expressing Flp recombinase. This method was successful in cloning a gene cluster responsible for lipopolysaccharide (LPS) modifications that confer polymyxin B resistance and in complementing its mutant. Further optimized procedures should be widely applicable because large insert fragments are precise clones of the wild-type genome.</p>
収録刊行物
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- The Journal of General and Applied Microbiology
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The Journal of General and Applied Microbiology 62 (5), 225-232, 2016
公益財団法人 応用微生物学・分子細胞生物学研究奨励会