Fig 1.
Structure and schematic behaviour of the LuxR-repressible promoters.
A) Schematic representation of the working principle of the repressible promoters. In the blue and red boxes the two synthetic networks representing the constitutive LuxR expression cassette and the LuxR-regulated RFP expression cassette are shown, respectively (curved green arrows represent promoters, purple ovals represent RBSs, coloured arrows represent genes and red hexagons represent transcriptional terminators). Green stars represent the AHL inducer molecules, diffusing through cell membrane and binding the LuxR transcription factor (blue circles). This complex represses the expression of RFP. B) The consensus sequence of the library of LuxR-repressible promoters is reported, according to the IUPAC nucleotide code (reported in the box on the right). -35 (red line), -10 (green line) and lux box (blue line) regions are highlighted. C) Generation method of the library via PCR with degenerate primers. The template is BBa_J23118 in the BBa_J61002 plasmid. In every box, the BioBrick code of the corresponding part is reported. The forward primer P_LUXH_FWD anneals with the template in a region falling between promoter and RBS and has a floating tail containing the degenerate sequence (the -35, lux box and -10 regions are highlighted in red, blue and green, respectively), while the reverse primer P_LUXH_REV anneals with the sequence downstream of the transcriptional terminator.
Table 1.
Parts and plasmid vectors used in this study.
Table 2.
List of oligonucleotides used to generate the library members and to sequence-verify all the plasmids.
Fig 2.
Characterization contexts for the library of LuxR-repressible promoters.
The 'constitutive mode' indicates the configuration in which the LuxR expression cassette is absent, while the 'regulated mode' indicates the context in which the LuxR expression cassette is present. A-B panels show the RFP-LC context, in constitutive and regulated mode, respectively; C-D represent the RFP-MC context, in constitutive and regulated mode, respectively; E-F represent the GFP-MC context, in constitutive and regulated mode, respectively. LC and MC plasmids are the pSB4C5 low-copy plasmid and pSB3K3 medium-copy plasmid, respectively. The '+' symbol in panel B indicates that both plasmids are present in the cell.
Fig 3.
Configurations studied to test the 'position effect' of the LuxR-expression cassette.
Here, the LuxR-repressible promoter BBa_J107100 (green curved arrow), driving the expression of RFP (red box) or GFP (green box), has been assembled in different configurations: in low-copy pSB4C5 (LC) plasmid with the RFP reporter gene A) without the LuxR-expression cassette, B) assembled downstream or C) upstream of the LuxR-expression cassette, and D) co-transformed with the LuxR-expression medium-copy plasmid pLuxR-MC; in medium copy pSB3K3 (MC) plasmid with the GFP reporter gene E) without the LuxR-expression cassette, F) assembled downstream or G) upstream of the LuxR-expression cassette. The BioBrick codes are indicated for each part and a name is provided for each configuration. The '+' symbol in panel D indicates that both plasmids are co-transformed in the cell.
Table 3.
Sequences of the promoters belonging to the LuxR-repressible promoter library.
Fig 4.
Constitutive activity of the LuxR-repressible promoters with three different measurement systems.
Bars represent the average of at least three experimental replicates and error bars represent the 95% confidence intervals. CV is reported when a statistically significant difference was measured among the three experimental conditions (p-value<0.05, ANOVA).
Fig 5.
Hill function parameter values obtained by fitting the repression curves of the promoters in the regulated mode.
Three different measurement systems are reported, as indicated on the figure.
Fig 6.
Single-cell measurements of the activity of the LuxR-repressible promoters.
A) Representative fluorescence distributions for BBa_J107100 promoter assayed in constitutive mode (green) and regulated mode with no (blue), intermediate (red) and full (cyan) induction. B) Promoter activity obtained via population-based measurements versus single-cell measurements. Blue circles represent average RPU values, horizontal and vertical error bars represent the 95% confidence intervals. The solid black line is the y = x line. C) Cell-to-cell variability, in terms of CV, is reported as a function of RPU.
Fig 7.
Boxplots showing the LuxR-repressible J107100 promoter activity in presence of a LuxR expression cassette in different positions.
The description of the tested contexts is provided in the text.