A practical challenge in using phage polymerases is that they can exhibit toxicity (20
). To overcome this, we constructed a variant of T7 RNAP with reduced toxicity by combining mutations to lower its concentration and activity. The wild-type gene was placed under control of an IPTG-inducible promoter in a low copy plasmid and co-transformed with a second plasmid containing a T7 promoter and fluorescent reporter (B). Toxicity was assessed by plating strains on inducing media and counting colonies after 24
h of growth. Significant toxicity was observed when T7 RNAP is highly expressed, even in the absence of a T7 promoter (C, v1). A Lon-mediated N
-terminal degradation tag from the umuD
gene in E. coli
) was added to limit polymerase concentration (C, v2). This resulted in a slightly lower toxicity. Next, RNAP expression was tightly controlled using a weak ribosome-binding site and GTG start codon. Finally, during cloning, a spontaneous mutation in the polymerase active site (R632S) arose. This mutation was found to significantly reduce host toxicity while maintaining activity (C, v3). It is interesting to note that the previous studies have reported that mutations to this region of the polymerase can reduce its processivity (32–34
Once the toxicity was reduced, we sought to expand the number of polymerases that are orthogonal and can be used simultaneously in a cell. In the context of a controller, this would increase the number of circuit outputs that could be linked to different pathways (A). Orthogonality can be achieved by engineering polymerases to bind to different promoters and not cross-react. One approach would be to apply part mining (35
), where homologous phage polymerases are identified from the sequence databases, constructed using DNA synthesis and screened for activity and orthogonality. However, we did not want to repeat the process of reducing the toxicity for each polymerase. To avoid this, we used the set of T7 backbones (v1 to v3) as scaffolds into which we inserted the peptide loop responsible for promoter recognition from different phage polymerases identified in the NCBI database (A). These chimeras were then tested for activity and orthogonality.
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’ (more ...)
The DNA sequence to which T7 RNAP binds is determined by a β-hairpin, known as the specificity loop, which contacts the −12 to −7
bp region of the promoter (36
). Changes to the specificity loop confer the ability to recognize different promoter sequences (37
). Remarkably, it had been shown that a single mutation (N748D) switches T7 RNAP to preferentially bind a T3-like phage promoter (39
). Thus, this is a good region to target mutagenesis; however, random mutations may be disruptive and it could require too many simultaneous mutations to generate orthogonality (40
). Instead, we took the approach of exchanging the entire β-hairpin identified in RNAP from homologous phages. This is related to previous work, where orthogonal transcription factors were made by using information from a sequence database to guide mutations to the protein-DNA interface (39
Each phage in the T7 family contains an RNAP and 10–20 promoters that provide a wealth of information about the interaction of the polymerase with DNA. To identify β-hairpins that confer different binding specificities, we identified homologues of T7 RNAP and computationally determined their DNA-binding preferences. First, 43 T7 RNAP homologues were identified from NCBI via a protein–protein BLAST against non-redundant protein sequences (25
). RNAP with an E-value less than 1−100
and for which a fully sequenced phage genome exists were selected for further analysis. A multiple sequence alignment of the RNAP amino acid sequences was performed using ClustalW (26
) to identify and extract the β-hairpin in each RNAP corresponding to T7 amino acids G732-P780. Three RNAPs (RSB2, W5455 and ϕIBB-PF7A) were eliminated due to significant differences in length of the β-hairpins. A second multiple sequence alignment was performed with only the β-hairpin sequences, and 13 RNAP subfamilies were identified (distance between members <
0.1 in the ClustalW guide tree). Putative promoters were identified from each phage genome using PHIRE, software that scans genomes for regulatory elements by identifying conserved sequences with a limited number of user-defined degeneracies (27
). WebLogo was used to determine the consensus sequence for each phage RNAP (41
). Remarkably, for each β-hairpin subfamily, the binding region of the consensus promoter is identical (Supplementary Figure S1
Novel RNAPs were generated by swapping the β-hairpin from T7 RNAP (Q744 to I761) with the equivalent region from each subfamily (B). The corresponding binding region of the T7 promoter (−12C to −7C) was replaced with the promoter subfamily consensus sequences (A). The resulting RNAPs were screened for activity against their predicted promoters. Four RNAPs exhibited strong activity (42-, 12-, 17- and 40-fold induction by T7*, T7*(T3), T7*(K1F) and T7*(N4), respectively) (Supplementary Figure S4
) and similar temporal induction (Supplementary Figure S6
). The activity of these RNAPs against non-cognate promoters was then characterized. Each RNAP is highly orthogonal, even after significant mutations to both the specificity loop and promoter (C).
It is valuable to be able to tune the strength of a promoter to achieve varied levels of transcription. The T7 promoter has been shown to be modular, consisting of a 6
bp RNAP-binding region and a 5
bp strength-determining region (36
). Mutations to the strength-determining region should alter promoter strength without affecting RNAP specificity (44
). We created a promoter library by randomizing the wild-type T7 promoter from −2bp to +3
bp. The library was screened using flow cytometry, and a set of representative promoters was identified that includes a broad range of expression levels spanning two orders of magnitude. These promoters were sequenced to determine the mutations to the strength-determining region (A). These regions were then combined with a different specificity-determining region that is specific for T7*(T3). The rank order for strength persists with fair correlation between strengths of individual promoters (B and Supplementary Figure S5
). Orthogonality is retained for the hybrid promoters, where T7* only transcribes those promoters containing the cognate-binding sequence and vice versa for T7*(T3).
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 (more ...)
The set of orthogonal RNAPs enables the independent control of multiple pathways by a genetic program encoded on a controller. The modularity of the controller allows it to be tested using fluorescent reporters before implementing it to control metabolic pathways or difficult to assay cellular functions. To demonstrate this, we constructed a simple genetic program whose output is two RNAPs (T7* and T7*(K1F)) under the control of multiple inducing signals and a logic gate (). T7* is expressed from the Ptac
promoter by IPTG induction (characterized in Supplementary Figure S3
), whereas T7*(K1F) is controlled by an AND gate that is active only in the presence of both IPTG and anhydrotetracycline (aTc). The AND function is achieved by placing a lacO-binding site after the transcription start site of the Ptet
promoter. Such promoter engineering has been applied previously to build gates (46
), and it is similar to that used to build an edge detector program (11
). We characterized the performance of the genetic program using reporter plasmids for each RNAP and verified that it produced the expected circuit logic (B). The reporters are placed under the control of promoters responsive to each RNAP in the same genetic context as the genes ultimately to be controlled. The T7* RNAP exhibits 7.8-fold induction, whereas the T7*(K1F) RNAP is induced 9.2-fold when tested using red fluorescent protein and a low copy pSC101* origin.
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 (more ...)
Once the controller is verified, it can then be co-transformed with the target pathways, which are carried on a second plasmid. We applied the genetic program to control the expression of two small molecule pigments, lycopene (red) and deoxychromoviridans (green). Escherichia coli
produces small amounts of lycopene through the 1-deoxy-d
-xylulose-5-phosphate (DXP) pathway following the introduction of the carotenoid genes crtEBI
). Lycopene production can be improved by overexpressing two genes, dxs
). Deoxychromoviridans is synthesized from l
-tryptophan by the genes vioABE
Figure 5. Control of multiple pathways using a genetic controller and orthogonal polymerases. (A) The controller described from is utilized to control production of two pigments (lycopene and deoxychromoviridans). Operons are drawn using SBOLv symbols ( (more ...)
Each gene from both pathways was placed in a cistron comprising an insulator, T7 or K1F promoter, synthetic ribosome-binding site, the gene of interest and a T7 terminator (A). A library of T7-derived terminators was created to avoid homologous recombination between cistrons (Supplementary Data, Supplementary Figures S8
). Synthetic cistrons were assembled into either a lycopene operon or a deoxychromoviridans operon using the Gibson assembly method (53
). This method enabled us to introduce a library of T7 promoters into each lycopene cistron, co-transform the library with T7*, and screen for efficient producers under inducing conditions. We identified and sequenced a clone that exhibited excellent pigment production and contrast (T7 promoters indicated in A). For deoxychromoviridans, we obtained sufficient expression using wild-type promoters but found that clones were not stable when vioE
was expressed as a cistron. Expressing vioB
from a single K1F promoter is stable and eliminates leaky expression of deoxychromoviridans.
We then assessed the feasibility of connecting the genetic program that was tuned and characterized in isolation to the multigene pigment pathways. The genetic program was co-transformed with the two biosynthetic pathways, and cells were plated on varying inducer combinations (C). Lycopene is synthesized in the presence of IPTG and is not affected by the presence of aTc. Deoxychromoviridans, in contrast, is only synthesized when both inducing molecules are present. Further quantification was performed by extracting pigments and measuring relative absorbance under the different inducing states (50
). We measured a 7.9-fold induction of lycopene expression between the absence of inducers and the addition of IPTG (D). Lycopene production in the presence of both IPTG and aTc yields a 5.6-fold induction, and induction of deoxychromoviridans is 3.3-fold. The circuit performance obtained using the fluorescent reporters () closely matches with that obtained using the more complex pathways (D).
Genetic engineering is becoming increasingly complex, requiring integrated control over multiple many-gene pathways. Ultimately, it is envisioned that synthetic systems could approach the size and complexity of complete genomes. Our ability to engineer systems at this scale is going to require modularization in their design and testing. To this end, we present an approach to decouple the genetic regulation controlling the conditions and dynamics of gene expression from the pathways that are being controlled. The sensors and circuits can be constructed and tuned using fluorescent proteins, and the engineered pathways can be optimized under simplified inducing conditions. Because the output channels are orthogonal polymerases, the ‘controller’ can switch from testing to implementation simply by co-transforming it with the more complex pathways. This has several advantages. First, the pathways may be challenging to assay and inappropriate for the characterization of circuit dynamics. Second, this approach enables the future development of highly integrated genetic programs linking dozens of sensors and circuits that all can be encoded in the controller. Thus, the modularization of genetic programming enables it to be abstracted from the idiosyncrasies of the biology being controlled.