Programming gene expression with combinatorial promoters

Abstract

Promoters control the expression of genes in response to one or more transcription factors (TFs). The architecture of a promoter is the arrangement and type of binding sites within it. To understand natural genetic circuits and to design promoters for synthetic biology, it is essential to understand the relationship between promoter function and architecture. We constructed a combinatorial library of random promoter architectures. We characterized 288 promoters in Escherichia coli, each containing up to three inputs from four different TFs. The library design allowed for multiple -10 and -35 boxes, and we observed varied promoter strength over five decades. To further analyze the functional repertoire, we defined a representation of promoter function in terms of regulatory range, logic type, and symmetry. Using these results, we identified heuristic rules for programming gene expression with combinatorial promoters.

Figure 1

Random assembly ligation generates a diverse promoter library. Promoters can be assembled out of modular sequence units. (A) The assembled sequence of an example promoter. The 5′ overhangs of each unit are shown in red. The RNA polymerase boxes (−10 and −35) are highlighted in yellow, and the predicted start site of transcription (+1) is capitalized. Operator colors are consistent throughout the figure. (B) Steps in promoter assembly and ligation into the luciferase reporter vector: promoters are assembled by mixed ligations using 1-bp or 2-bp cohesive ends, and then ligated into a luciferase reporter plasmid. (C) Luminescence measurements in 16 inducer conditions (±each of four inducers, as indicated) for the promoter shown in (A). The output levels determine promoter logic. Note that this promoter does not respond to LuxR regulation at the distal region. (D) The 48 unique units used in the library contain operators responsive to the four TFs (indicated by color) in the regions distal, core, and proximal (Sequences in Supplementary Table S1). The promoter fragments corresponding to (A) are boxed in red.

Figure 2

Activation functions at distal and is attenuated by intrinsic promoter strength. (A) Measurements of promoters activated at distal operators. These promoters respond only to LuxR (solid triangles) or AraC (open triangles) induction. Some promoters fail to respond even though they contain a functional operator (points on the solid line). The activation ceiling (red dashed line) represents the maximal observed activation and does not depend on the unregulated expression level. (B) Promoters containing operators at core (squares) or proximal (circles) do not respond to induction.

Figure 3

Repression is effective at all three positions, following the trend core⩾proximal⩾distal. Measurements of repressed single-input promoters. Responses are colored according to the repressor: LacI (filled) or TetR (open). Each promoter contains a single operator located at distal (A), core (B), or proximal (C) positions. Single-input activities are plotted in the induced (unregulated) versus uninduced (repressed) states. In some promoters, operators do not effectively repress the promoter (points located near solid black line). Luciferase detection limits are shown with gray dashed lines.

Figure 4

Dual-input gates exhibit diverse functions in logic-symmetry space. Promoter response phenotypes can be represented by their asymmetry, a (y-axis), logic type, l (x-axis), and regulatory range, r. (A) Diagram showing the space of allowed logical phenotypes, with the locations of ideal logic gates indicated. The SIG responds completely to one inducer and not at all to the other. The SLOPE gate represents an intermediate logical function between AND and OR, whereas the asymmetric gates are intermediate between SIG and the corresponding symmetric gate. Intermediate logical behavior is represented between these ideal locations. The logic-symmetry parameterization is defined in the Materials and methods. Points outside of the dashed triangle are not accessible if promoters respond monotonically to each input. (B) The logical phenotypes of 50 dual-input promoters (r>3). AR promoters are shown as purple circles, RR promoters are shown as gold disks. The diameter of each disk is proportional to the logarithm of its regulatory range, r.

Figure 5

Combinatorial promoter architecture reveals rules for programming gene expression. The architecture and function of several dual-input promoters. The architecture of each promoter (colored according to Figure 1) is shown with its functional operators and −10 and −35 boxes. (A) RR promoters respond to both LacI and TetR. The fourth induction column (+ IPTG, +aTc) corresponds to the unregulated state. (B) AR promoters respond to AraC and one of the two repressors, as indicated. Here, the third column (+IPTG/aTc, −Lara) corresponds to the unregulated state.

Figure 6

The distribution of operator locations in natural promoters reflects functional trends of synthetic promoters. Operator locations are as annotated in RegulonDB 5.0 (Salgado et al, 2006). Distributions of repressor (A) and activator (B) operators as found in 1102 E. coli promoters. The number of operators centered at each position relative to the start site of transcription (+1) is plotted. (C) The density of operators found in 554 σ⁷⁰ promoters is broken down into three promoter regions, distal, core, and proximal, as well as regions upstream (5′ remote) and downstream (3′ remote) of the promoter. The density is shown as the fraction of sites in each position weighted by the relative size (bp) of each region.