Multiple large segment deletion method for Corynebacterium glutamicum |
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Authors: | Nobuaki Suzuki Hiroshi Nonaka Yota Tsuge Satoshi Okayama Masayuki Inui Hideaki Yukawa |
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Institution: | (1) Microbiology Research Group, Research Institute of Innovative Technology for the Earth (RITE), 9-2, Kizugawadai, Kizu-Cho, Soraku-Gun, Kyoto 619-0292, Japan;(2) Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan |
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Abstract: | A precise and scarless genome excision method, employing the Cre/loxP system in concert with double-strand break (DSB)-stimulated intramolecular recombination was developed. The DSBs were mediated by the restriction endonuclease, I-SceI. It permitted multiple deletions of independent 14-, 43-, and 10-kb-long genomic regions on the Corynebacterium glutamicum genome. Accuracy of deletion was confirmed by the loss of marker genes, PCR, and sequencing of new genome joints. Eleven, 58, and 4 genes were predicted on the 14-, 43-, and 10-kb deleted regions, respectively. Although the resultant mutant lost a total of 67 kb encoding 73 genes, it still exhibited normal growth under standard laboratory conditions. Such a large segment deletion method in which multiple, successive deletions are possible is useful for genome engineering. |
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