Expression vectors for Acinetobacter baylyi ADP1

Abstract

Acinetobacter baylyi ADP1 is naturally competent and proficient at homologous recombination, so it can be transformed without restriction digests or ligation reactions. Expression vectors for this system, however, are not yet widely available. Here we describe the construction and characterization of inducible expression vectors that replicate as plasmids in A. baylyi or integrate into a nonessential part of its chromosome. These tools will facilitate the engineering of genes and genomes in this promising model organism.

Fig 1

Examples of plasmid (top) and integration (bottom) vectors. Each contains a single BioBrick flanked by restriction sites EcoRI-NotI-XbaI (prefix) and SpeI-NotI-PstI (suffix). The BioBrick in the integration vector (bottom) was assembled from smaller BioBricks carrying the flanks, expression cassette (regulator-promoter-reporter gene), and selectable marker. The vectors rely upon the function of foreign promoters and ribosome binding sites (each pair is indicated by an open triangle) in A. baylyi ADP1.

Fig 2

Novel expression vectors function in A. baylyi ADP1. Wild-type A. baylyi ADP1 cells were transformed with plasmid and integration vectors (described in Table 1). Each transformed strain was separately propagated to mid-log phase and challenged with an inducer (IPTG, arabinose, or p-hydroxybenzoate) or water (negative control) for 3 h. The cells were harvested by centrifugation and lysed; the cell extract was reacted with BMUG, and the formation of the fluorescent 4-methylumbelliferone product was monitored in a microtiter plate spectrofluorimeter. The activity was derived from the slope (fluorescence per unit of time) and plotted on the log scale. Error bars represent standard errors from three independent trials.