A plasmid toolbox for controlled gene expression across the Proteobacteria

Abstract

Controlled gene expression is fundamental for the study of gene function and our ability to engineer bacteria. However, there is currently no easy-to-use genetics toolbox that enables controlled gene expression in a wide range of diverse species. To facilitate the development of genetics systems in a fast, easy, and standardized manner, we constructed and tested a plasmid assembly toolbox that will enable the identification of well-regulated promoters in many Proteobacteria and potentially beyond. Each plasmid is composed of four categories of genetic parts (i) the origin of replication, (ii) resistance marker, (iii) promoter-regulator and (iv) reporter. The plasmids can be efficiently assembled using ligation-independent cloning, and any gene of interest can be easily inserted in place of the reporter. We tested this toolbox in nine different Proteobacteria and identified regulated promoters with over fifty-fold induction range in eight of these bacteria. We also constructed variant libraries that enabled the identification of promoter-regulators with varied expression levels and increased inducible fold change relative to the original promoter. A selection of over 50 plasmids, which contain all of the toolbox's genetic parts, are available for community use and will enable easy construction and testing of genetics systems in both model and non-model bacteria.

Figure 1.

Plasmid toolbox assembly scheme and nomenclature. Each plasmid is composed of four genetic parts that share overlapping primer sequences, requiring only four primer pairs to assemble any version of the plasmid. Plasmids are named based on the codes provided, in the order: origin, regulator, reporter and marker.

Figure 2.

Experimental outline and induction screen results. (A) Workflow for inducible systems screens. (B) Promoter-regulators with >50-fold induction range. Fold change was calculated without correcting for autofluorescence of the cells and medium. Floating lines represent the induction range of mRFP with fluorescence in the absence of inducer plotted at the bottom of each line and induced expression plotted at the top of the vertical line. Data is clustered by the host strain. Strains on x-axis: Pa (P. aeruginosa), Pp (P. putida), Af (A. fabrum), Bt (B. thailandensis), Xc (X. campestris), Ab (A. baylyi), Sb (Sulfitobacter sp. EE-36) and Rp (Ruegeria sp. TM1040). Horizontal lines at each cluster represent the average fluorescence of control strains that did not possess mRFP. (C) Fold Change Heatmap of all Bacteria and Inducible Systems. The fold change was calculated from RFU data normalized to OD and background fluorescence of the medium and empty vector control after 24 h of growth for all bacteria except A. fischeri, where the medium only was used for normalization. Strain abbreviations are the same as in (B) plus Av (A. fischeri). Inducible systems on the y-axis are labeled with the transcription factor. For TetR systems, TetR-1 refers to TetR/P_TetA and TetR-2 refers to TetR/P_Ltet-O1.

Figure 3.

Induction range of 12 expression systems in nine Proteobacteria. (A) Expression of mRFP for each of 12 inducible expression systems after overnight growth in each bacterium with induction range represented by floating bars with fluorescence in the absence of inducer plotted at the bottom of each bar and induced expression plotted at the top. (B) Expression of mRFP in late-exponential phase of growth graphed by the inducible system. On each graph, expression from the nine Proteobacteria are displayed in the following order: P. aeruginosa, B. thailandensis, A. fabrum, P. putida, A. baylyi, X. campestris, Ruegeria sp. TM1040, Sulfitobacter sp. EE-36, and A. fischeri. Data is presented without correcting for autofluorescence of the cells and medium.

Figure 4.

Measurement of mRFP at Titrated Inducer Concentrations. (A) pFLxR5 in P. putida (B) pKNR5 in X. campestris (C) pBCyR5 in P. aeruginosa. Vertical bars represent range of expression at five concentrations of inducer in RFU and gray circles are OD₆₆₀ at late stationary phase ± SE of triplicates. (D) pFCiR5 in A. fabrum (E) pFAR5 in B. thailandensis (F) pBLtR5 in Ruegeria sp. TM1040. Data points represent fluorescence normalized to growth (OD₆₆₀) from samples grown in the absence of inducer (U) and at five inducer concentrations. Exponential phase (closed circles) and late stationary phase (open squares) ± SE of triplicates. (G) Expression data from independent induction experiments. Strains containing two plasmids with unique promoter-regulator pairs and reporters were induced both individually and simultaneously. For each bacterium, the top and bottom graphs show fluorescence data from GFP and mRFP, respectively. Plasmid combinations are listed in Supplementary Table S6. For each data cluster, floating lines represent expression from the following conditions in order: expression with inducer for GFP (closed circle), expression with inducer for mRFP (closed circle), and expression with both inducers (open square). Data from strains with the corresponding single plasmid are included on mRFP graphs (dashed line). The data shown are the average RFU of triplicates after an overnight induction.

Figure 5.

Conditionally essential gene to measure tightness of repression. The gentamicin acetyltransferase gene aacC1 is placed under control of P_CymRC in non-inducing and inducing conditions. (A) B. thailandensis pKCyGe2 strains are plotted as a percentage of the total number of viable cells containing the plasmid. (B) Serial dilutions of B. thailandensis plated onto media containing gentamicin or the backbone antibiotic kanamycin with and without the addition of the inducer cumate. Data from cultures in exponential phase of growth.

Figure 6.

Expression of Total Library and Select Library Isolates. Expression data from all screened isolates of the LuxR/P_LuxB library in P. aeruginosa (top left), A. fabrum (top right), and A. fischeri (center left), the TetR/P_TetA library in B. thailandensis (center right) and A. fabrum (bottom left), and the NahR^AM/P_SalTTC library in A. fabrum (bottom right). Grey and black lines show uninduced and induced expression of each isolate, respectively, and the overlayed scatterplot shows corresponding fold change. Symbols in red represent the fold change of original plasmids. Data is sorted by induced RFU. Inserted floating bar charts represent expression ranges from isolates with the highest fold change from each respective library. Expression range from original plasmid represented in a shaded box, fluorescence from empty vector control shown as a black horizontal line. Data is an average of three replicates after overnight induction.