Herrero M, de Lorenzo V, Ensley B, Timmis K N
Centro de Investigaciones Biológicas CSIC, Madrid, Spain.
Gene. 1993 Nov 30;134(1):103-6. doi: 10.1016/0378-1119(93)90181-2.
A general method to construct recombinant Pseudomonas putida (and related bacteria), which transcribe specific genes inserted into their chromosome in response to the presence of alkyl- and halobenzoates, has been developed. The system is based on the ability of the T7 RNA polymerase (T7pol) to initiate transcription from cognate promoter sequences located upstream from cloned genes. A specialized transposon, mini-Tn5 xylS/Pm::T7pol, was constructed which contains the structural T7 gene 1 downstream from the XylS protein/benzoate-regulated Pm promoter of the meta-operon of the TOL catabolic plasmid. This transposon was stably inserted into the chromosome of a P. putida target strain so that gene 1 is transcribed upon exposure of the bacteria to benzoate effectors of the XylS regulator. Genes whose expression is to be mediated by T7pol are cloned in mini-Tn5 transposons containing T7 promoter sequences upstream from the cloning site and then the hybrid transposons are inserted into different positions in the same chromosome. In this way, expression of the genes cloned within the mini-Tn5 vectors is dependent on the T7pol/XylS/Pm system. This expression device is particularly well suited for applications in which the expression of two or more genes is to take place in response to a single chemical signal, i.e., exposure to certain aromatic compounds.
已开发出一种构建重组恶臭假单胞菌（及相关细菌）的通用方法，该方法可使插入其染色体中的特定基因在烷基苯甲酸酯和卤代苯甲酸酯存在时进行转录。该系统基于T7 RNA聚合酶（T7pol）从克隆基因上游同源启动子序列起始转录的能力。构建了一种特殊的转座子mini-Tn5 xylS/Pm::T7pol，它在TOL分解代谢质粒的间位操纵子的XylS蛋白/苯甲酸酯调节的Pm启动子下游含有结构T7基因1。该转座子稳定插入恶臭假单胞菌目标菌株的染色体中，使得当细菌暴露于XylS调节因子的苯甲酸酯效应物时基因1得以转录。其表达由T7pol介导的基因被克隆到mini-Tn5转座子中，这些转座子在克隆位点上游含有T7启动子序列，然后将杂种转座子插入同一染色体的不同位置。通过这种方式，mini-Tn5载体中克隆基因的表达依赖于T7pol/XylS/Pm系统。这种表达装置特别适用于两个或更多基因的表达要响应单一化学信号（即暴露于某些芳香化合物）的应用。
