Construction of chromosomally encoded lacZ and gfp reporter strains of Escherichia coli for the study of global regulation of metabolism

Abstract

We developed plasmid systems pJLIC and pJLRCS that allow the easy cloning of promoter regions upstream of reporter genes (lacZ and gfp) and the subsequent transfer of the resulting reporter cassettes into the chromosome of Escherichia coli by recombineering. Thus, a promoter region can be inserted upstream of lacZ gene in the context of the lactose operon of recipient E. coli strains. Moreover, a promoter–GFP reporter is inserted at a nonessential site of the chromosome. These systems allow for selection and promoter strength analysis at the single copy level. We also provide a collection of E. coli knock‐out mutants in genes for global regulation of C, N, and O metabolism, and combinations of these mutants with the newly constructed chromosomal lacZ and gfp reporter system. This allows assaying transcriptional activities of promoters before the background of different mutants. Both lacZ and gfp reporter cassettes (including appropriate antibiotic resistance markers) and the regulator knock‐outs may be easily transferred by phage P1 mediated transduction to other E. coli strains. As a proof of principle, ß‐galactosidase activity and GFP fluorescence were compared for lacZ and gfp reporter systems placed under the control of the PrssA promoter both in a wild‐type and an isogenic rpoS‐mutant strain.