CONSTRUCTION OF A CASSETTE ENABLING REGULATED GENE-EXPRESSION IN THE PRESENCE OF AROMATIC-HYDROCARBONS

A high-level expression cassette has been constructed from a TOL plasmid derived fromPseudomonas putida carrying allcis- andtrans-acting regulatory elements necessary for transcriptional gene activation in the presence of aromatic hydrocarbons such as toluene. Foreign DNA can be inserted at unique KpnI, SacI, andEcoRI sites 7, 13, and 15 nucleotides downstream of a ribosome binding site. The cassette, flanked by BamHI and EcoRI restriction sites, was inserted into a broad-host-range vector and its efficacy demonstrated in various purple bacteria by monitoring the expression of a reporter gene spectrophotometrically and by SDSPAGE. High-level induction (80- to 600-fold) was detected inEnterobacteriaceae and inPseudomonas but was absent or low inAgrobacterium tumefaciens andRhizobium leguminosarum. © 1992 Academic Press, Inc. All rights of reproduction in any form reserved.