Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.
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.
Guzman
L M
LM
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Belin
D
D
Carson
M J
MJ
Beckwith
J
J
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics; D051777:AraC Transcription Factor; D001089:Arabinose / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004790:Enzyme Induction; D004794:Enzyme Repression; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015971:Gene Expression Regulation, Enzymologic; D005804:Genes, Lethal / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000235:genetics; D014157:Transcription Factors
D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D001089:Arabinose; D017853:Phosphotransferases (Alcohol Group Acceptor); C022550:ribulokinase; D000469:Alkaline Phosphatase