AN IMPROVED SYSTEM FOR GENE REPLACEMENT AND XYLE FUSION ANALYSIS IN PSEUDOMONAS-AERUGINOSA
AN IMPROVED SYSTEM FOR GENE REPLACEMENT AND XYLE FUSION ANALYSIS IN PSEUDOMONAS-AERUGINOSA
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DOI：
10.1016/0378-1119(95)00055-b
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发表时间：
1995-05-26
期刊：
影响因子：
3.5
通讯作者：
HOANG, TT
中科院分区：
文献类型：
--
作者：
SCHWEIZER, HP;HOANG, TT
构建了一种新的基于pUC 19的基因置换载体。该载体掺入（i）反向选择性sacB标记，（ii）lacZ α。用于蓝白筛选的等位基因，（iii）用于缀合介导的质粒转移的oriT，和（iv）用于SmaI和稀有切割大范围核酸酶I-SceI的独特克隆位点。这些罕见的限制性位点也存在于辅助质粒pUC 19 Sce上。替换载体被改造成含有很少的限制性位点，以获得更多的克隆DNA片段内的限制性位点，从而促进其遗传操作。通过将新构建的xylE：：Gm（R）融合盒整合到铜绿假单胞菌的glpD基因中，证明了该系统的有用性。
A novel pUC19-based gene replacement vector has been developed. This vector incorporates (i) the counterselectable sacB marker, (ii) a lacZ alpha. allele for blue-white screening, (iii) an oriT for conjugation-mediated plasmid transfer and (iv) unique cloning sites for SmaI and the rare-cutting meganuclease I-SceI. These rare restriction sites are also present on the helper plasmid pUC19Sce. The replacement vector is engineered to contain few restriction sites to gain greater access to restriction sites within cloned DNA fragments, thus facilitating their genetic manipulation. The usefulness of the system was demonstrated by chromosomal integration of a newly constructed xylE::Gm(R) fusion cassette into the glpD gene of Pseudomonas aeruginosa.