A bacteriophage T7-based expression vector, pBT7, with color selection for the recombinant

Abstract

A bacterial expression plasmid, pBT7, based on a bacteriophage T7 RNA polymerase/promoter system, has been devised for positive color selection of clones containing an insert. The T7 gene 10 promoter and its N-terminal portion, 89 nucleotides in length, have been amplified by the polymerase chain reaction and cloned into the multiple cloning site of plasmid pUC18 in the opposite direction to lacZ'. This insertion does not inactivate the lacZ alpha-peptide activity. Hence, bacteria carrying pBT7 without an insert form blue colonies on an indicator plate, while bacteria carrying recombinant clones with an insert appear as white colonies. Using pBT7, part of the Caenorhabditis elegans unc-13 gene product has been overproduced at a high level, approaching 50% of total Escherichia coli protein.