A new Thermus-Escherichia coli shuttle integration vector system
A new Thermus-Escherichia coli shuttle integration vector system
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DOI：
10.1128/jb.179.15.4811-4814.1997
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发表时间：
1997-08-01
影响因子：
3.2
通讯作者：
Oshima, T
中科院分区：
文献类型：
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作者：
Tamakoshi, M;Uchida, M;Oshima, T
我们建立了一株完全缺失pyrE基因（编码嘧啶生物合成途径的角化磷酸核糖基转移酶）的嗜热热菌。我们还构建了一个整合载体，由大肠杆菌质粒载体pBluescript和嗜热T. leu操纵子序列的2.1 kb片段组成，用于将外源基因整合到嗜热T. leu的染色体上。以pyrE和leuB基因为探针，对整合载体进行检测。携带pyrE基因的整合载体pINV通过单次交叉事件以6 × 10（-5）的频率转化Delta pyrE菌株。leuB基因也可以作为整合载体系统的另一个标记。向量可以在期望的位置积分。用一种独特的限制性内切酶消化嗜热t菌转化体的染色体DNA，体外循环后将载体恢复到大肠杆菌中。卡那霉素核苷酸转移酶基因可通过pINV在嗜热菌中成功表达。
We established a Thermus thermophilus strain in which the pyrE gene (coding for orotate phosphoribosyltransferase of the pyrimidine biosynthetic pathway) was totally deleted. We also constructed an integration vector, which consisted of the Escherichia coli plasmid vector pBluescript and a 2.1-kb segment of the T. thermophilus leu operon sequence, for the integration of a foreign gene into a chromosome of the thermophile. pyrE and leuB genes were used as probes to test the integration vector. The integration vector pINV, bearing the pyrE gene, transformed the Delta pyrE strain at a frequency of 6 x 10(-5) through a single crossover event. The leuB gene could also be used as another marker of the integration vector system. The vector could be integrated at the expected site. By digesting the chromosomal DNA of the T. thermophilus transformants with a unique restriction enzyme, the vector could be recovered into E. coli after the recircularization in vitro. The kanamycin nucleotidyltransferase gene could be successfully expressed in the thermophile by using pINV.