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 synthbuetic constitutive promoters upstream of RFP, with a broad host range BBR1 origin and a kanamycin resistance marker. The family also has seven inducible promoters (Jungle Express, NahR, XylS, RhaS, Lac, LacUV5, and TetR) controlling RFP expression on BBR1/kanamycin plasmid backbones. For four of these inducible promoters (Jungle Express, NahR, Lac, and 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.

Identification of novel broad host-range promoter sequences functional in diversePseudomonadotaby a promoter-trap approach

“…5), which was not an expected outcome. This phenomena might be related to a strain-dependent behavior, as some iGEM teams reported differences in the promoter strength of Anderson promoters when assessed in E. coli DH5α 39 evaluated all Anderson promoters in E. coli XL1blue, finding also some discrepancies to the original description. Considering the same example from above, expression from J23113 was higher (1.2-fold increase) to that from J23100 40 .…”

Assessing horizontal gene transfer in the rhizosphere ofBrachypodium distachyonusing fabricated ecosystems (EcoFABs)

“…The images of the inoculated roots were taken using EVOS M7000 fluorescence microscope (Thermo Fisher Scientific, Waltham, MA) using objectives with 2X and 40X magnification. For imaging purposes, we replaced the plasmid in the donor strain with p101mNeonGreen [30] with constitutively expressed neon green fluorescence. Both donor (green) and recipient (mCherry red) were inoculated in the B. distachyon rhizosphere grown for 2 weeks and images were taken 24 hours post-inoculation.…”

Genetically refactoredAgrobacterium-mediated transformation

“…To better understand how to control bacterial gene expression within plants, we evaluated the activity of 16 synthetic constitutive, and 4 inducible promoters in bacterial cells grown in rich media 40 , as well as infiltrated into the leaf tissue of Nicotiana benthamiana and Arabidopsis thaliana. In both plants, bacterial constitutive promoter activity correlated highly between leaf tissues, and between observed in vitro activity (Figure 1A, Figure S1).…”

“…All other compounds unless otherwise specified were purchased through Sigma Aldrich. Bacterial kinetic growth curves were performed as described previously 40 .…”

“…Characterization of pGinger vectors harbored by A. fabrum in vitro was performed as previously described for other bacteria 40 . Briefly, A. fabrum C58C1 with different pGinger vectors were grown overnight in 10mL of LB supplemented with kanamycin overnight at 30°C with 250 rpm shaking and then diluted 1:100 into 500 μL of fresh LB media with kanamycin in a deep-well 96-well plate (Corning) For inducible promoters, chemical inducers were added in two-fold dilutions before incubation.…”