The pGinger Family of Expression Plasmids

Abstract

The pGinger suite of expression plasmids comprises 43 plasmids that will enable precise constitutive and inducible gene expression in a wide range of Gram-negative bacterial species. Constitutive vectors are composed of 16 synthetic constitutive promoters upstream of red fluorescent protein (RFP), with a broad-host-range BBR1 origin and a kanamycin resistance marker. The family also has seven inducible systems (Jungle Express, Psal/NahR, Pm/XylS, Prha/RhaS, LacO1/LacI, LacUV5/LacI, and Ptet/TetR) controlling RFP expression on BBR1/kanamycin plasmid backbones. For four of these inducible systems (Jungle Express, Psal/NahR, LacO1/LacI, and Ptet/TetR), we created variants that utilize the RK2 origin and spectinomycin or gentamicin selection. Relevant RFP expression and growth data have been collected in the model bacterium Escherichia coli as well as Pseudomonas putida. All pGinger vectors are available via the Joint BioEnergy Institute (JBEI) Public Registry. IMPORTANCE Metabolic engineering and synthetic biology are predicated on the precise control of gene expression. As synthetic biology expands beyond model organisms, more tools will be required that function robustly in a wide range of bacterial hosts. The pGinger family of plasmids constitutes 43 plasmids that will enable both constitutive and inducible gene expression in a wide range of nonmodel Proteobacteria.

Keywords: molecular genetics; plasmid; synthetic biology.

FIG 1

Plasmid architecture of the pGinger suite. The pGinger plasmids share a naming convention in which the first two letters after “pGinger” correspond to the origin and resistance marker, respectively, followed by the expression system. All plasmids share the same architecture as in the map of pGingerBK-TetR, whereby a conserved RBS-RFP is downstream of the promoter, followed by a strong terminator. All selectable markers are upstream of the promoter, with the origin between the marker and the RFP cassette.

FIG 2

Activity of constitutive promoters in E. coli and P. putida. RFP expression normalized to cell density from Anderson promoters within either E. coli (y axis) or P. putida (x axis) is shown with standard deviations (n = 3). The background fluorescence of the two bacteria is indicated by “WT” (wild type). Optical density measurements are shown in Fig. S1 in the supplemental material. AU, arbitrary units.

FIG 3

Activity of inducible systems in E. coli and P. putida. RFP expression normalized to cell density (y axis) from inducible systems within either E. coli (top panel in orange) or P. putida (bottom panel in blue) is shown as a function of millimolar concentration of inducer (x axis). Fits to the Hill equation are shown as dashed lines and shaded to show confidence intervals. Raw data points are overlaid (n = 3). Corresponding optical density measurements are shown in Fig. S2.

FIG 4

Activity of inducible pGinger variants in E. coli. For origin and selection marker pGinger variants of Jungle Express (top left), LacO1/LacI (top right), Psal/NahR (bottom left), and pTet/TetR (bottom right), dose-response curves of normalized RFP expression are shown as a function of millimolar concentration of inducer. Error bars represent standard deviations (n = 3). Note that the x axis is nonlinear. Corresponding optical density measurements are shown in Fig. S3. Results of kinetic experiments for these systems are shown in Fig. S4 to S7.