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. 2012 Sep 1;40(17):8773-81.
doi: 10.1093/nar/gks597. Epub 2012 Jun 28.

Modular control of multiple pathways using engineered orthogonal T7 polymerases

Karsten Temme  1 , Rena Hill, Thomas H Segall-Shapiro, Felix Moser, Christopher A Voigt
Affiliations

Modular control of multiple pathways using engineered orthogonal T7 polymerases

Karsten Temme et al. Nucleic Acids Res. .

Abstract

Synthetic genetic sensors and circuits enable programmable control over the timing and conditions of gene expression. They are being increasingly incorporated into the control of complex, multigene pathways and cellular functions. Here, we propose a design strategy to genetically separate the sensing/circuitry functions from the pathway to be controlled. This separation is achieved by having the output of the circuit drive the expression of a polymerase, which then activates the pathway from polymerase-specific promoters. The sensors, circuits and polymerase are encoded together on a 'controller' plasmid. Variants of T7 RNA polymerase that reduce toxicity were constructed and used as scaffolds for the construction of four orthogonal polymerases identified via part mining that bind to unique promoter sequences. This set is highly orthogonal and induces cognate promoters by 8- to 75-fold more than off-target promoters. These orthogonal polymerases enable four independent channels linking the outputs of circuits to the control of different cellular functions. As a demonstration, we constructed a controller plasmid that integrates two inducible systems, implements an AND logic operation and toggles between metabolic pathways that change Escherichia coli green (deoxychromoviridans) and red (lycopene). The advantages of this organization are that (i) the regulation of the pathway can be changed simply by introducing a different controller plasmid, (ii) transcription is orthogonal to host machinery and (iii) the pathway genes are not transcribed in the absence of a controller and are thus more easily carried without invoking evolutionary pressure.

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Figures

Figure 1.
Figure 1.
Separation of synthetic regulation onto a genetic controller using orthogonal phage polymerases. (A) The controller contains genetic sensors and circuits, the output of which is the expression of a phage RNAP, which then activates a pathway. The pathways can be programmed to respond to different conditions by swapping the controller without further genetic manipulation. (B) Modifications are shown to reduce the toxicity of T7 RNAP, the details of which are described in the text. (C) Toxicity of T7 RNAP variants is shown. Each variant was co-transformed with a different plasmid bearing a T7 promoter, and cells were grown on plates containing 1 mM IPTG to fully induce RNAP expression from the Ptac promoter. RNAP variants containing different combinations of modifications are shown (v1 = T7 RNAP in pIncW plasmid, v2 = addition of Lon tag to v1, v3 = addition of weak RBS and GTG start codon to v2). The data are shown for the expression of the T7 variant in the absence of a T7 promoter (black, plasmid N23, Supplementary Data) and the T7 promoter driving red fluorescent protein (white, plasmid N155, Supplementary Data). Asterisks indicate that no colonies were observed.
Figure 2.
Figure 2.
Design of orthogonal T7 RNAPs and promoters. (A) Sequences of the specificity loop and promoter are shown for each orthogonal polymerase. (B) The interaction between the T7 RNAP and T7 promoter is shown (36). The β-hairpin ‘specificity’ loop (red) interacts with the 5′ end of the promoter in the region marked ‘binding’. (C) The orthogonality between the engineered RNAP promoter pairs is shown. Each RNAP was co-transformed individually with each promoter cloned into the base plasmid N155 (Supplementary Data) and expressed by 1 mM IPTG induction. The data represent the average of three experiments performed on different days after subtraction of the autofluorescence of Escherichia coli harbouring no plasmid. The standard deviation of the experiments is reported in Supplementary Figure S4 and representative cytometry distributions are shown in Supplementary Figure S7.
Figure 3.
Figure 3.
Modularity of T7 promoters. (A) A library of pT7 promoters of differing strengths was created by mutating the strength-determining region (−2bp to +3bp) and cloned into plasmid N155 (Supplementary Data). Synonymous mutations were also made to the pT3 promoter. Sequences are shown for the two libraries. (B) The promoter libraries were used to control fluorescent reporter proteins, and each library was co-transformed with both T7* and T7*(T3). Activity of co-transformants under 1 mM IPTG induction was characterized by flow cytometry to study cognate interactions (T7*-PT7, top left and T7*(T3)-PT3, bottom right) and non-cognate interactions (T7*-PT3, bottom left and T7*(T3)-PT7, top right). Error bars represent the standard deviation of three experiments on different days.
Figure 4.
Figure 4.
Design and characterization of a two-input genetic program. (A) A genetic program is shown that controls two orthogonal polymerases via different logic. The activity of T7* was measured by co-transformation with a PT7 reporter plasmid (N489, Supplementary Data) with different combinations of inducer (1 mM IPTG and/or 10 ng/ml aTc). (B) K1F* activity was measured using a PK1F fluorescent protein reporter (THSS40, Supplementary Data, 1 mM IPTG and/or 10 ng/ml aTc). Error bars represent the standard deviation of three experiments of different days.
Figure 5.
Figure 5.
Control of multiple pathways using a genetic controller and orthogonal polymerases. (A) The controller described from Fig. 4 is utilized to control production of two pigments (lycopene and deoxychromoviridans). Operons are drawn using SBOLv symbols (55): RBSs (half circles), insulators (crossed circles) and terminators (T). SynBERC Registry part numbers (registry.synberc.org) are shown for each part, and nucleotide numbers for the plasmids are indicated. T7 promoter strength is reported as the fluorescence of a RFP reporter from plasmid N155 (Supplementary Data) and presented in relative expression units (REU, Supplementary Data, Supplementary Figure S2). (B) Biosynthetic pathways for lycopene and deoxychromoviridans are shown. Intermediate products are shown in black, and genes expressed by the engineered system are shown in italics. DMAPP, dimethylallyl diphosphate; DXP, 1-deoxy-d-xylulose 5-phosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl pyrophosphate; G3P, glyceraldehyde 3-phosphate; HMBPP, (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate; IPA, indole-3-pyruvic acid; IPP, isopentenyl diphosphate. (C) Pigment production follows the logic encoded by the controller. Escherichia coli strain MG1655 was co-transformed with plasmids encoding the genetic program and both pathways. Cells were grown overnight in non-inducing conditions, diluted to an OD600 of 1.0, and spotted onto plates containing inducers. In the presence of IPTG and aTc, both pigments are synthesized. The combination of green and red pigments appears brownish purple. Photographs of representative colonies are shown. (D) Quantitative assessment of pigment production. Cells were grown for 24 h in LB media containing the indicated inducers. Lycopene and deoxychromoviridans were extracted, and absorbance was measured (lycopene, 580 nm; deoxychromoviridans, 650 nm). Data are reported as the fraction of maximum absorbance observed. Error bars represent standard deviation of three experiments of different days.
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