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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >IMPROVED BROAD-HOST-RANGE RK2 VECTORS USEFUL FOR HIGH AND LOW REGULATED GENE EXPRESSION LEVELS IN GRAM-NEGATIVE BACTERIA
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IMPROVED BROAD-HOST-RANGE RK2 VECTORS USEFUL FOR HIGH AND LOW REGULATED GENE EXPRESSION LEVELS IN GRAM-NEGATIVE BACTERIA

机译：改良的广谱寄主RK2矢量，可用于革兰氏阴性细菌的高和低调控基因表达水平

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This report describes the construction and use of improved broad-host-range expression vectors based on the previously constructed pJB137 and pJB653 plasmids (Blatny et al., 1997). These vectors contain the minimal replicon of RK2 and the inducible Pu or Pm promoters together with their regulatory xylR or xylS genes, respectively, from the Pseudomonas putida TOL plasmid pWWO. A set of ATG vectors were derived from pJB653, and these vectors are characterized by the relatively small size, the presence of multiple cloning sites downstream of Pm, the establishment of their nucleotide sequence, the presence of RK2 oriT, and different antibiotic selection markers. The copy numbers of all the vectors can easily be modified by using copy-up mutations of the trfA gene, required for initiation of replication of RK2 replicons. The vectors were used to study the expression levels of the Acetobacter xylinum phosphoglucomutase gene celB and the two commonly used reporter genes luc and car in Escherichia coli, Pseudomonas aeruginosa, and Xanthomonas campestris. Good induction properties and tight regulation of Pm were achieved in all three species tested, and higher gene expression levels were obtained by using the ATG vectors compared to pJB653. By introducing different trfA copy-up mutations into the vectors, a wide range of gene expression levels from Pu and Pm were obtained in E. coli. Induced expression levels of luc, cat, and celB from Pm were found to be comparable to or higher than those from the Ptrc and P-T7 promoters located on high copy number plasmids. The induced levels of Luc activity were higher in P. aeruginosa than in E. coli, indicating that these vectors may be useful for maximization of gene expression in strains other than E. coli. We believe that the well-characterized vectors described here are useful for gene expression studies and routine cloning experiments in many Gram-negative bacteria. (C) 1997 Academic Press. [References: 68]
机译：该报道描述了基于先前构建的pJB137和pJB653质粒的改进的宽宿主表达载体的构建和使用（Blatny等，1997）。这些载体分别包含来自恶臭假单胞菌TOL质粒pWWO的RK2和可诱导的Pu或Pm启动子的最小复制子以及它们的调节性xylR或xylS基因。一组ATG载体衍生自pJB653，这些载体的特征是相对较小，在Pm下游存在多个克隆位点，其核苷酸序列的建立，RK2 oriT的存在以及不同的抗生素选择标记。通过使用启动RK2复制子复制所需的trfA基因的复制突变，可以轻松地修改所有载体的拷贝数。使用该载体研究木醋杆菌磷酸葡萄糖突变酶基因celB以及两个常用的报告基因luc和car在大肠杆菌，铜绿假单胞菌和樟脑黄单胞菌中的表达水平。与pJB653相比，在所有三个被测物种中均实现了良好的诱导性能和对Pm的严格调控，并且使用ATG载体获得了更高的基因表达水平。通过将不同的trfA复制突变引入载体，在大肠杆菌中获得了来自Pu和Pm的多种基因表达水平。发现来自Pm的luc，cat和celB的诱导表达水平与位于高拷贝数质粒上的Ptrc和P-T7启动子的诱导水平相当或更高。在铜绿假单胞菌中诱导的Luc活性水平高于在大肠杆菌中的Luc水平，表明这些载体可用于最大化除大肠杆菌之外的菌株中的基因表达。我们认为，此处描述的特征明确的载体可用于许多革兰氏阴性细菌的基因表达研究和常规克隆实验。 （C）1997学术出版社。 [参考：68]

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