Larsen J E, Albrechtsen B, Valentin-Hansen P
Nucleic Acids Res. 1987 Jul 10;15(13):5125-40. doi: 10.1093/nar/15.13.5125.
We describe the construction of low copy number operon-fusion vectors, and use one of these vectors for the cloning and transcriptional analysis of the terminator region after the deo operon of Escherichia coli K-12. The new vectors are miniderivatives of plasmid R1 containing the parB stability locus of this plasmid and the lac genes as a selectable marker. Since the copy number of the vectors is only one per genome-equivalent at temperatures below 37 degrees C this system is ideally suited for isolation and characterization of transcriptional and translational signals from E. coli. Our results show that a very strong terminator (deot), which resembles Rho-independent terminators, is located 60 bp downstream from the fourth structural gene of the deo operon. This confirms that deoD is the last gene in the operon. In addition, we have identified a new promoter just after the deot terminator and a short DNA sequence that is able to reduce lacZ expression by 85% when inserted between the deoP2 promoter and the lac genes.
我们描述了低拷贝数操纵子融合载体的构建，并使用其中一种载体对大肠杆菌K-12的deo操纵子之后的终止子区域进行克隆和转录分析。新载体是质粒R1的微型衍生物，含有该质粒的parB稳定性位点和作为选择标记的lac基因。由于在低于37摄氏度的温度下，载体的拷贝数仅为每个基因组当量一个，因此该系统非常适合于分离和表征来自大肠杆菌的转录和翻译信号。我们的结果表明，一个非常强的终止子（deot），类似于不依赖Rho的终止子，位于deo操纵子第四个结构基因下游60 bp处。这证实了deoD是该操纵子中的最后一个基因。此外，我们在deot终止子之后鉴定出一个新的启动子，以及一个短DNA序列，当插入deoP2启动子和lac基因之间时，能够使lacZ表达降低85%。
