Design, construction and characterization of a set of insulated bacterial promoters

Abstract

We have generated a series of variable-strength, constitutive, bacterial promoters that act predictably in different sequence contexts, span two orders of magnitude in strength and contain convenient sites for cloning and the introduction of downstream open-reading frames. Importantly, their design insulates these promoters from the stimulatory or repressive effects of many 5'- or 3'-sequence elements. We show that different promoters from our library produce constant relative levels of two different proteins in multiple genetic contexts. This set of promoters should be a useful resource for the synthetic-biology community.

Figure 1.

Comparison of promoter organization. Schematic of an insulated promoter (A) and a minimal uninsulated promoter (B). The promoter recognition region (PRR) containing the −10 and −35 RNAP binding determinants is shown in light gray, the transcription-initiation site (+1) is represented by arrow. In the insulated promoter, the surrounding genetic context (dark gray) is separated from the PRR by insulation sequences (diamond-filled pattern). Most elements known to effect transcription initiation and promoter escape are contained within the insulated promoter cassette boundaries. Because of its smaller size, the genetic context surrounding the minimal promoter is more likely to contain sequences that can effect transcription initiation, thereby increasing the possibility of context-dependent activity. (C) GFP synthesis rates per cell were measured for a control construct lacking GFP, a minimal promoter (j23101) or an insulated promoter (proD).

Figure 2.

Promoter strength. The strength of each promoter was measured in triplicate in E. coli DH5α grown in minimal media using GFP as a reporter (14). The GFP-synthesis rate is reported on a log₁₀ scale as a surrogate of promoter strength. Promoter-strength measurements relative to proD are listed in Table 1.

Figure 3.

Effect of sequences inserted 5′ or 3′ of the promoter. (A) The UP sequence was cloned upstream of either insulated (proA, proB, proC, proD) or uninsulated (j23113, j23150, j23151, j23101) promoters and promoter strength was measured using the GFP-reporter assay. Promoter strength was normalized to the strength of proD resulting in RPU. The inset shows relative strength of each promoter with the UP sequence normalized to the strength of the parental promoter. A value of 1 indicates no change in promoter strength. The promoter j23113 was excluded from comparative analysis due to its weak promoter strength and relatively large colony-to-colony variation. (B) The ‘anti’ sequence was cloned downstream of either insulated or uninsulated promoters and the promoter strength of each construct was measured using the GFP-reporter assay. For each promoter that could be robustly measured, the inset reports the ratio of promoter strength with the anti sequence to that without the insertion.

Figure 4.

Aparent promoter activities driving production of GFP versus Gemini or dsRed. (A) Protein-synthesis rates for GFP (top), Gemini (left) or dsRed (right) were determined by measuring fluorescence and using Equation (1). (B) Relative promoter strength was calculated from the protein-synthesis rates for a set of insulated (left) or uninsulated (right) promoters driving the production of GFP (x-axis) or Gemini (y-axis). To allow comparison between open-reading frames, each synthesis rate was normalized to that of proD driving production of the same open-reading frame [Equation (2)]. (C) Promoter strength was measured as described above for either GFP (x-axis) or dsRed (y-axis). Because production of dsRed from proD was decreased, each synthesis rate was normalized to that of j23101 driving production of the same open reading frame.

Figure 5.

Promoter activity from a chromosomal locus. Promoters driving the expression of GFP were recombined onto the chromosome downstream of the tonB locus, and the activity of the promoter was measured using GFP fluorescence. (A) GFP synthesis rate per cell. (B) Plots correlating the relative promoter activity either from the plasmid, pSB3C5 or the chromosome (at the tonB locus) for the insulated promoters (left) or the uninsulted promoters (right).