A versatile multiple- and single-copy vector system for the in vitro construction of transcriptional fusions to lacZ

Abstract

A multiple-copy (plasmid) vector and a single-copy (lambda) vector were constructed for the in vitro formation of transcriptional fusions to lacZ. In both vectors the transcription of lacZ is dependent upon the attachment of a promoter upstream, but beta-galactosidase is independently translated from the hybrid mRNA. These vectors are based on the W205 trp-lac deletion, but most of the trpBA DNA has been removed. Promoters are fused to the 3' end of trpA rather than the HindIII site at the 5' end of trpB used in other vectors containing the W205 deletion. This modification avoids the polar effects encountered when either an untranslated sequence, or a sequence translated in a different reading frame is fused to trpB. Hence the level of beta-galactosidase synthesized by fusions in these new vectors accurately reflects the frequency of transcription from the attached promoter. A polyrestriction site linker precedes the lacZ gene in both vectors and allows the direct ligation of promoter containing DNA fragments produced by a large collection of restriction endonucleases.