The pGinger Family of Expression Plasmids

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. 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.

Still, given that many bacteria require very particular combinations of promoters, origins, and selectable markers to enable controlled gene expression, there remains a need for vectors that will allow rapid prototyping of genetic circuits in understudied bacteria.
To facilitate the exploration of nonmodel hosts, we have developed a small suite of plasmids that permit both constitutive and inducible expression from the broad-host-range origin of replication BBR1 using a kanamycin selection marker. For a subset of the inducible systems that are known to work across multiple hosts, we have assembled combinatorial variants that utilize the compatible broad-host-range origin RK2 (14) as well as both spectinomycin and gentamicin selection markers. This family of plasmids, which we have named the pGinger suite, requires no assembly of these parts, can be easily cloned into via standard Gibson assembly techniques, and has both digital sequences and physical samples that can be publicly accessed through the Joint BioEnergy Institute (JBEI) registry (15).

RESULTS
Design and architecture of pGinger plasmids. All pGinger vectors express red fluorescent protein (RFP) with a consensus ribosomal binding site (RBS; TTTAAGAAGGAGATATACAT) derived from the BglBrick plasmid library. The overall conserved plasmid architecture and naming convention of the pGinger suite are shown in Fig. 1.
The BBR1 origin and kanamycin resistance cassette of relevant pGinger vectors were both derived from plasmid pBADTrfp (16). To develop a family of constitutive expression plasmids, the AraC coding sequence and promoter of pBADTrfp were replaced with 16 different synthetic promoters from the Anderson Promoter Library (http://parts.igem.org/ Promoters/Catalog/Anderson). For the inducible vectors, the AraC coding sequence and promoter of pBADTrfp were replaced with the following seven inducible systems: Jungle Express, derived from pTR_sJExD-rfp (17); Psal/NahR, derived from pPS43 (18); Prha/RhaS, derived from pCV203 (18); Ptet/TetR, derived from pBbE2a-RFP (6); Pm/XylS, derived from pPS66 (18); LacO1/LacI, derived from pBbE6a-RFP (6); and LacUV5/LacI, derived from pBbE5a-RFP (6). Three of these inducible systems, Pm/XylS, Psal/NahR, and Prha/RhaS, utilize an activator or bifunctional transcription factor; the other systems feature transcriptional repressors. These BBR1 vectors contain the mob element that facilitates conjugal transfer. For four of the inducible systems (Jungle Express, Psal/NahR, LacO1/LacI, and Ptet/TetR), additional vectors were constructed that varied both the origin and antibiotic marker. All RK2 origins were derived from pBb(RK2)1k-GFPuv (8), while the gentamicin resistance cassette was derived 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. from pMQ30 (19) and the spectinomycin resistance cassette was derived from pSR43.6 (20). The RK2 vectors do not contain the mob element. A full description of each pGinger vector can be found in Table 1.
Evaluation of constitutive expression pGinger plasmids. To evaluate the relative strength of constitutive Anderson promoters in the context of the pGinger vectors, plasmids were introduced into both Pseudomonas putida and E. coli. Fluorescence was measured after growth in lysogeny broth (LB) medium for 24 h. When fluorescence was normalized to cell density, expression from Anderson promoters showed significant correlation (Spearman's r = 0.49; P = 0.045) between P. putida and E. coli (Fig. 2). Promoters J23103 and J23113 were significantly stronger in E. coli than in P. putida, while promoter J23111 was significantly stronger in P. putida. Promoter sequences and mean expression values in both E. coli and P. putida are listed in Table 2.
Evaluation of inducible pGinger plasmids. The expression of the seven inducible systems within the pGinger suite was evaluated using the BBR1 origin and kanamycin marker (pGingerBK) against a titration of the inducer in both E. coli and P. putida (Fig. 3). All systems  showed inducibility in E. coli, and all but the rhamnose-inducible system Prha/RhaS showed inducibility in P. putida. Relevant expression characteristics of the inducible pGingerBK vectors in both tested bacteria are listed in Table 3. The strongest normalized expression from an inducible system in E. coli was the Ptet/TetR system, while both the strongest in P. putida were found to be Psal/NahR and Jungle Express inducible systems, which showed nearly identical  a For each Anderson promoter, the sequence is provided as well as the mean cell density-normalized RFP fluorescence in both E. coli and P. putida. Standard deviations are provided in parentheses (n = 3). The background fluorescence of E. coli is indicated by "WT" (wild type).
The pGinger Family of Expression Plasmids Microbiology Spectrum maximal expression. In both bacteria, the Jungle Express system demonstrated the greatest level of induction relative to background expression.
To evaluate the effects of various origin and selectable markers on expression from inducible systems, all six variants of Jungle Express, LacO1/LacI, Psal/NahR, and Ptet/TetR were investigated for their dose responses to their inducer molecules in E. coli (Fig. 4). Relevant expression parameters are listed in Table 4. In general, BBR1 variants showed greater expression than RK2 origin plasmids, which was expected given the higher copy number of BBR1 plasmids in E. coli (11). Among the pGinger Jungle Express vectors, both pGingerRS-JE and pGingerRK-JE showed dose responses distinct from those of the other vectors (Fig. 4, top left). Notably, all pGingerRS (RK2-spectinomycin) plasmids showed the lowest expression across each system tested (Table 4).

DISCUSSION
The pGinger suite of plasmids offers researchers an array of small, preassembled vectors that will permit rapid identification of useful genetic elements in diverse Gram-negative bacteria due to the use of broad-host-range origins (RK2) and selectable markers known to work across many species (kanamycin, spectinomycin, and gentamicin). The compatibility of RK2 and BBR1 origins may also permit researchers to introduce multiple pGinger vectors into a single

The pGinger Family of Expression Plasmids
Microbiology Spectrum strain simultaneously (14). In combination with other recent plasmid suites that have been publicly released, the pGinger plasmids have the potential to facilitate more advanced synthetic biology and metabolic engineering efforts in bacterial species that have been traditionally understudied. a For each inducible system on a BBR1 origin with a kanamycin marker, the experimentally observed background (uninduced) fluorescence and maximal fluorescence are given for both E. coli and P. putida. Standard deviations are provided in parentheses (n = 3). Additionally, the inducer concentration used to achieve maximal expression and the relative induction levels are listed. Max, maximum. Plasmid design and construction. All plasmids were designed using Device Editor and Vector Editor software, while all primers used for the construction of plasmids were designed using j5 software (15,21,22). Plasmids were assembled via Gibson assembly using standard protocols (23). Plasmids were routinely isolated using the Qiaprep Spin miniprep kit (Qiagen, USA), and all primers were purchased from Integrated DNA Technologies (IDT; Coralville, IA). The fluorescent protein used in all plasmids was mRFP1 (24).
Characterization assays. To characterize RFP expression from the vectors, we measured optical density and fluorescence after growth in 96-well plates for 24 h. First, overnight cultures were inoculated into 5 mL of LB medium from single colonies and grown at 30°C or 37°C. These cultures were then diluted 1:100 into 500 mL of LB medium with the appropriate antibiotic in 96 square v-bottom deep-well plates (Biotix DP22009CVS). For characterization of the inducible systems, inducer was added to wells in the first column of the plate at the maximum concentration tested and diluted 2-fold across the plate until the last column, which was left as the zero-inducer control. Plates were sealed with a gas-permeable microplate adhesive film (Axygen BF400S) and cultures were grown for 24 h at either 30°C or 37°C with shaking at 200 rpm. Optical density was measured at 600 nm, and fluorescence was measured at an excitation wavelength of 535 nm and an emission wavelength of 620 nm. All data were analyzed and visualized using custom Python scripts using the SciPy (25), NumPy (26), Pandas, Matplotlib, and Seaborn libraries. Fits to the Hill equation were done as previously described (27).
Plasmid and sequence availability. All strains and plasmid sequences from Table 1  between Lawrence Berkeley National Laboratory and the U.S. Department of Energy. The views and opinions of the authors expressed herein do not necessarily state or reflect those of the U.S. Government or any agency thereof. Neither the U.S. Government nor any agency thereof, nor any of their employees, makes any warranty, expressed or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan