Molecular Cloning of the Escherichia coli Glutamine Permease Operon, glnP, Using Mini-F Plasmid
The genes encoding the Escherichia coli high-affinity transport system for glutamine, glutamine permease operon, were cloned by means of phenotypic complementation using a mini-F cloning vehicle. Plasmid pTN101 contains the structural gene for the periplasmic glutamine binding protein, glnH, and at least one other gene, glnP, also required for the permease activity. It is difficult to reclone the entire operon into high-copy number plasmids pBR322 or pUC18: The resultant plasmids are extremely unstable and are rapidly lost from the host cell. Subcloning of the operon in pBR322 resulted in the loss of Gln+ phenotype, an ability to utilize glutamine as a sole source of carbon. Direct cloning of the permease operon by using pBR322 was unsuccessful; although three types of Gln+ growers were obtained, they do not contain DNA in the glnP locus.
