Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in gram-negative bacteria
Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in gram-negative bacteria
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DOI：
10.1006/plas.1997.1294
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发表时间：
1997-01-01
期刊：
影响因子：
2.6
通讯作者：
Valla, S
中科院分区：
文献类型：
--
作者：
Blatny, JM;Brautaset, T;Valla, S
该报告描述了基于先前构建的pJB 137和pJB 653质粒的改进的宽宿主范围表达载体的构建和使用（Blatny等人，1997年）。这些载体含有来自恶臭假单胞菌TOL质粒pWWO的RK 2的最小复制子和诱导型Pu或Pm启动子以及它们的调节xylR或xylS基因。一组ATG载体衍生自pJB 653，并且这些载体的特征在于相对小的尺寸、Pm下游的多克隆位点的存在、它们的核苷酸序列的建立、RK 2 oriT的存在以及不同抗生素选择标记。所有载体的拷贝数可以通过使用trfA基因的拷贝突变容易地进行修饰，这是RK 2复制子复制起始所需的。用该载体研究了木醋杆菌磷酸葡萄糖变位酶基因celB和两个常用的报告基因luc和car在大肠杆菌、铜绿假单胞菌和野油菜黄单胞菌中的表达水平。良好的诱导性能和严格的调节Pm在所有三个物种测试，并获得更高的基因表达水平，通过使用ATG载体相比，pJB 653。通过在载体中引入不同的trfA拷贝突变，在E.杆菌发现来自Pm的luc、cat和celB的诱导表达水平与来自位于高拷贝数质粒上的Ptrc和P-T7启动子的诱导表达水平相当或更高。铜绿假单胞菌的Luc活性诱导水平高于大肠杆菌。表明这些载体可用于在除大肠杆菌以外的菌株中最大化基因表达。杆菌我们相信，这里描述的良好表征的载体是有用的基因表达研究和常规克隆实验在许多革兰氏阴性菌。(C)北京：科学出版社.
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.