Guzman L M, Belin D, Carson M J, Beckwith J
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Bacteriol. 1995 Jul;177(14):4121-30. doi: 10.1128/jb.177.14.4121-4130.1995.
We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.
我们构建了一系列质粒载体（pBAD载体），其包含阿拉伯糖（araBAD）操纵子的PBAD启动子以及编码该启动子正负调节因子的araC基因。利用phoA基因和phoA融合体来监测这些载体中的表达，我们发现，与基于PTAC的载体的50倍相比，诱导/抑制比可达1200倍。phoA表达可在很宽的诱导剂（阿拉伯糖）浓度范围内调节，并且由于葡萄糖的存在而降低至极低水平，葡萄糖可抑制表达。此外，诱导和抑制的动力学非常迅速，并且受到宿主菌株中ara等位基因的显著影响。因此，使用这个能够高效且快速开启和关闭的系统，可以以非常简单的方式且无需改变温度来研究细菌生理学的重要方面。我们利用PBAD启动子的严格调控来研究必需基因的无效突变的表型，并探索了将pBAD载体用作表达系统的用途。
