Fig 1.
hsvtk::aph fusion gene as an ON-/OFF-selector.
(A) The construct for the expression of hsvTK::APH. The reading frames of hsvTK (excluding stop codon, 376 aa) and APH (excluding the start codon, 263 aa) were fused without a linker, resulting in hsvtk::aph. The fusion gene was placed under the T5 promoter (pT5) with the translation initiation site (rbs score [25], 13843). (B) OFF-selection using hsvTK::APH selector. E. coli MG1655 harboring either a plasmid expressing hsvTK::APH or a plasmid expressing GFPUV (negative control) were incubated with dP (0–1,000 nM), and the number of viable (colony forming) cells was measured after 3 h incubation. (C) ON-selection. E. coli MG1655 harboring either of the plasmids were treated with Km (0–200 μg/mL), and the number of viable cells was measured after 3 h incubation. The bar heights show the average of 3 samples, and the error bars indicate the standard deviation. Asterisks indicate that no colony was observed.
Fig 2.
hsvtk::cat fusion gene as a ON/OFF-selector.
(A) The construct for the expression of hsvTK::CAT. The reading frames of hsvTK and CAT (excluding the start codon, 217 aa) were fused without a linker, resulting in hsvtk::cat. The fusion gene was placed under the T5 promoter (pT5) with the translation initiation site (rbs score [25], 13843). (B) OFF-selection. E. coli MG1655 harboring either a plasmid expressing hsvTK::CAT or a plasmid expressing GFPUV (negative control) were incubated with dP (0–1,000 nM), and the number of viable (colony forming) cells was measured after 3 h incubation. (C) ON-selection. E. coli MG1655 harboring either of the plasmids were treated with Cm (0–240 μg/mL), and the number of viable cells was measured after 3 h incubation. The bar heights show the average of 3 samples, and error bars indicate the standard deviation.
Fig 3.
Selection of functional lux switches from lux box libraries.
(A) Library design. Part (six nucleotides) of the lux box (LuxR-binding sites) was randomized by PCR mutagenesis, yielding Library-1 and -2. The fusion selector hsvtk::aph, together with sfgfp, was placed under the promoter library. LuxR was constitutively expressed from a different plasmid. (B) Selection procedure: first, approximately 107 transformant cells were cultured for several hours in the presence of 1 μM dP (OFF-selection). Next, the cells were treated with 50–200 μg/mL Km (ON-selection) cultured for 1–6 hours in the presence of 1 μM 3OC6-HSL. The resultant cells were rinsed and re-grown in LB media. The cell pool was subjected to the flow cytometry in each step of this selection. (C) Flow cytometric analysis of the transformant pools before selection and after OFF/ON-selection (incubated in the absence/presence, respectively, of 1,000 nM 3OC6-HSL). Histograms in color indicate the fluorescent distribution in the presence of 3OC6-HSL, while those in gray indicate the fluorescent distribution in the absence of 3OC6-HSL.
Fig 4.
Dose-response of lux promoter variants to 3OC6-HSL activated by LuxR.
Five individual lux promoter variants (#30, #08, #17, #01, and #05) were selected from 45-randomly picked ones from the pool shown in S1 Fig. (C) (variants recovered from OFF-selected Library-2 that survived the ON selection (incubation with 50 μg/mL of Km for 4 h)) and quantitatively characterized using micro-titer plate measurement of sfGFP expression after 12 h growth in medium containing various concentrations of 3OC6-HSL (0–105 nM). The bar heights show the average of 3 samples, and the error bars indicate the standard deviation.