Strict regulation of gene expression from a high-copy plasmid utilizing a dual vector system.
High-copy plasmids are useful for producing large quantities of plasmid DNA, but are generally inadequate for tightly regulating gene expression. Attempts to suppress expression of genes on high-copy plasmids often results in residual or "leaky" production of protein. For stringent regulation of gene expression, it is often necessary to excise the gene of interest and subclone it into a low-copy plasmid. Here, we report a dual plasmid technique that enables tight regulation of gene expression driven by the lac promoter in a high-copy vector. A series of plasmids with varying copies of the lacI(q) gene have been constructed to permit titration of the LacI protein. When a high-copy plasmid is transformed along with the appropriate lacI(q)-containing plasmid, tight gene regulation is achieved, thus eliminating the need to subclone genes into low-copy plasmids. In addition, we show that this dual plasmid technique enables high-copy gene expression of a protein lethal to Escherichia coli, the ccdB protein. In principle, this technique can be applied to any high-copy plasmid containing the popular pUC replication of origin and provides an easier means of obtaining rigid control over gene expression.
Gruber
David F
DF
Brown University, Department of Psychiatry and Human Behavior, Division of Biology and Medicine, 171 Meeting Street, Room 187, Box G-B187, Providence, RI 02912, USA. David_Gruber@Brown.edu
Pieribone
Vincent A
VA
Porton
Barbara
B
Kao
Hung-Teh
HT
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D007763:Lac Operon; D010957:Plasmids / Q000235:genetics*
D029968:Escherichia coli Proteins