Improved single and multicopy lac-based cloning vectors for protein and operon fusions

Abstract

We describe several new vectors for the construction of operon and protein fusions to the Escherichia coli lacZ gene. In vitro constructions utilize multicopy plasmids containing suitable cloning sites located between upstream transcription terminators and downstream lac operon segments whose lacZ genes retain or lack translational start signals. Single-copy λ prophage versions of multicopy constructs can be made genetically, without in vitro manipulation. The new vectors, both single and multicopy, are improved in that they have very low levels of background lac gene expression, which makes possible the easy detection and accurate quantitation of very weak transcriptional and translational signals. These vectors were developed for analysis of the expression of IS 10's transposase gene, which is transcribed less than once per generation, and whose transcripts are translated on average less than once each. Both single and multicopy constructs can also be used to select mutations affecting fusion expression, and mutations isolated in single-copy constructs can be crossed genetically back onto multicopy plasmids for further analysis.