Tight regulation, modulation, and high-level expression by vectors containing the arabinose P(BAD) promoter

Abstract

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 tight regulation of the P(BAD) promoter to study the phenotypes of null mutations of essential genes and explored the use of p(BAD) vectors as an expression system.