TIGHT REGULATION, MODULATION, AND HIGH-LEVEL EXPRESSION BY VECTORS CONTAINING THE ARABINOSE P-BAD PROMOTER
TIGHT REGULATION, MODULATION, AND HIGH-LEVEL EXPRESSION BY VECTORS CONTAINING THE ARABINOSE P-BAD PROMOTER
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DOI：
10.1128/jb.177.14.4121-4130.1995
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发表时间：
1995-07-01
影响因子：
3.2
通讯作者：
BECKWITH, J
中科院分区：
文献类型：
--
作者：
GUZMAN, LM;BELIN, D;BECKWITH, J
我们构建了一系列质粒载体（pBAD载体），其含有araBAD（阿拉伯糖）操纵子的P-BAD启动子和编码该启动子的正调控子和负调控子araC的基因。使用phoA基因和phoA融合来监测这些载体中的表达，我们表明诱导/阻遏的比率可以是1，200倍，而基于P-TAC的载体为50倍。phoA的表达可以在诱导物（阿拉伯糖）浓度的宽范围内调节，并通过抑制表达的葡萄糖的存在降低至极低水平。此外，诱导和阻遏的动力学非常迅速，并受到宿主菌株中ara等位基因的显著影响。因此，可以有效和快速地打开和关闭的该系统的使用允许以非常简单的方式并且在不改变温度的情况下研究细菌生理学的重要方面。我们利用P-BAD启动子的光调控研究了必需基因无效突变的表型，并探索了pBAD载体作为表达系统的用途。
We have constructed a series of plasmid vectors (pBAD vectors) containing the P-BAD 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 P-TAC-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 light regulation of the P-BAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.