Introduction

Engineering synthetic gene circuits entails the redesign of existing gene networks or the creation of novel genetic functions to perform a predetermined task. Construction of these circuits has been valuable in attaining a bottom up understanding of biological systems[1] and offers potential for harnessing biological function for biotechnology[2,3] and biomedicine[4,5]. Well-characterized genetic components have been integrated into circuits that function as logic gates [6,7], memory elements, clocks[8] and counters. Ultimately, like their electronic analogues, components can be assembled into larger networks for practical applications in medicine, bioremediation[9] and chemical synthesis [10]. However, despite the fact that a rapidly growing number of gene circuits are being published, the complexity of the systems is not keeping pace[11]. One factor in this apparent complexity barrier is that gene circuits typically operate within cellular systems and rely on the host’s endogenous promoters and translational machinery to drive circuit function. Therefore, unintended interactions with endogenous processes can make implementation of predictable and rationally designed circuits difficult. Consequently, orthogonal expression systems that insulate the synthetic gene circuits from other biological networks are required[12-14].

One such orthogonal expression system exploits the mono-subunit T7 RNA polymerase, which is commonly utilized because of its simplicity, stability, processivity, and specificity. In contrast to multi-subunit bacterial RNA polymerases, T7 polymerase recognizes a specific 17 base pair promoter sequence and does not require co-factors to activate transcription [15]. T7 RNA polymerase is also highly processive and has been extensively used for achieving high yield protein expression[16,17]. Furthermore, since T7 RNA polymerase is highly specific for T7 promoters, its use permits exclusive expression from user-defined genes in a variety of different backgrounds without interference from endogenous promoters[18]. For these reasons, T7 promoters have been used in protein expression applications in both live cells and in cell-free systems. Therefore, T7 circuit elements will be valuable not just in cell based systems for achieving orthogonal expression but can also be used in cell-free systems to assemble circuits of increasing complexity [19-22].

However, a significant challenge to implementing complex gene regulatory circuits with T7 promoters is that, unlike bacterial promoters, the commonly used viral promoters have few natural mechanisms for activating or repressing gene expression. Synthetic T7 promoter variants can be repressed by transcription factors such as LacI and TetR only when bound to a relatively short regulatory region positioned proximal to and downstream from the transcription start site [16,23-27]. As a consequence of the short cis-regulatory region, engineering T7 promoters that respond to multiple transcriptional regulators is very challenging and few strategies regulation of the T7 promoter by multiple transcription factors have been described [28].

In this article, we develop an approach for engineering T7 promoters that can be regulated by multiple inputs, and we use this approach to implement a digital logic function. To achieve this, we first focused on extending the effective regulatory region of the T7 promoter. As an alternative to cis-regulation of T7 promoter, we harness DNA looping to enable regulation from distal locations. In natural systems, DNA looping can be mediated by protein multi-merization and is commonly used to enable transcription regulation by the synergistic action of repressors bound at different locations. For instance, Lac repressor proteins (LacI) bind their operators as a tetramer or a dimer of dimers. Native E. coli lac promoters contain auxiliary Lac operators (LacO) upstream and downstream to the E. coli lac promoter. At low LacI concentrations, presence of additional Lac operators induces DNA loop formation in the intervening DNA, thereby increasing the probability of LacI occupancy of the E. coli lac promoter [29,30] and enhancing repression from E. coli promoters. We show that, as with E. coli lac promoters, an improvement in repression of T7lacO promoters can be attained by appropriately spacing a second lac operator 92 bases upstream to a T7lacO promoter. We then demonstrate that this extension of the T7 regulatory region through DNA looping enables the development of multi-input regulation. Specifically, the binding of a second regulator, TetR, at a distal site interferes with the loop enhanced repression of the first regulator, LacI. We examine the effect of placement of tetO, the binding site for the TetR protein, into the LacI looping framework so as to generate T7 promoters that respond to both TetR and LacI (Figure 1). We found that Tet operators placed in between the two lac operators interfere with Lac mediated repression.

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Figure 1. Design strategy for achieving combinatorial regulation of expression from T7 promoters.

A) An auxiliary lacO is placed upstream to a conventional T7lacO promoter to create stronger LacI repressible T7 promoters. DNA looping is induced by the binding of a single LacI tetramer to both of the lacO binding sites. B) TetR binding regions (tetO) placed within this DNA looping framework, at regions indicated by grey box, can enable multi-input regulation by interfering with LacI mediated looping.

https://doi.org/10.1371/journal.pone.0078442.g001

Using our dual-input T7 promoters, we present an extendable strategy for creating T7-based logic gates. We demonstrate an IMPLIES logic gate which accepts TetR and LacI proteins as inputs. IMPLIES gates together with NOT gates can be used to perform any kind of logic operation, thereby providing a way for implementing logic functions of choice from T7 promoters[31]. Furthermore, we demonstrate the functionality of these TetR and LacI repressible T7 promoters in both live E. coli cells and in cell-free expression systems.

Materials and Methods

GFP measurements from E. coli experiments

Enhanced GFP (eGFP)[32] expressing plasmids bearing different T7lacO and tet operator regions were co-transformed along with pTetRLacI plasmids into BL21-AI cells. A single colony from the transformation plate was used to initiate an overnight culture in LB media. A small aliquot of overnight culture was then transferred into M9 minimal media supplemented with 100 µg/mL ampicillin and 50 µg/mL kanamycin. The culture was incubated at 37°C for ~5 hours before this starter culture was again diluted in M9 media to a final optical density (OD₆₀₀) of 0.01. 0.02% L-arabinose was added to the culture to induce the expression of T7 RNA polymerase. 100 µL aliquots of culture were dispensed into a 96-well plate (Corning 3370). Subsequently, IPTG and aTc were added to the wells as indicated. 50 μl of mineral oil was added to each of these wells to prevent drying of the samples. Absorbance (at 600 nm wavelength) and fluorescence measurements (485/20 nm excitation, 528/20 nm emission) were made at intervals of 7 minutes in a BioTek Synergy 2 plate reader. Fluorescence values indicated in Figures 2 and 3 represent measurements made at 200 minutes. Fluorescence values were corrected for background fluorescence of the media and cells in the absence of L-arabinose. Absorbance readings at 600 nm were used to normalize for cell density. Error bars in all the graphs represent standard deviation from three technical replicates.

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Figure 2. Effect of auxiliary operators on LacI mediated repression of T7lacO promoters (in vivo).

A) illustrates promoter sequences containing T7lacO promoters with auxiliary operator sequences of different strengths. B) Protein expression responses to 30 μM IPTG from the constructs depicted in A). GFP concentration units are expressed as µM/OD₆₀₀. C) Dose responses to IPTG from the different constructs. Fluorescence response values are normalized to cell counts as determined by optical density values. Error bars depict standard deviation of triplicate measurements. Lines depict nonlinear regression fits to the Hill equation.

https://doi.org/10.1371/journal.pone.0078442.g002

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Figure 3. Effect of tetO on LacI mediated repression of T7lacO when tetO is in between the two lac operators (in vivo).

Shown in (A) are the plasmid constructs pDRT7 14 and pDRT7 77. B) Displays the responses of these plasmids to presence /absence of 30 μM IPTG and 200 ng/ml aTc. C) Gene expression response, as determined by the normalized fluorescence response, of the pDRT7 77 plasmid to a range of IPTG and aTc concentrations. aTc concentration (ng/mL) is displayed on the X axis and the Y-axis denotes IPTG concentrations (μM). GFP fluorescence measurements in B and C are expressed as µM/OD₆₀₀. D) is a schematic of the IMPLIES logic gate realized using the pDRT7 77 plasmid. Error bars depict standard deviation of triplicate measurements.

https://doi.org/10.1371/journal.pone.0078442.g003

Cell-free expression experiments

Qiagen EasyXpress Protein Synthesis Kits were used for carrying out cell-free protein synthesis reactions. This kit was chosen because it does not contain IPTG and therefore would not interfere with the testing of the promoters designed in this study. In addition, the extract supplies LacI in concentrations sufficient for repression of the T7lacO promoter based constructs. Reactions were set up following the manufacturer’s instructions, but the final reaction volume was scaled down to 15 μL. The reactions were overlaid with 10 μL mineral oil to prevent drying. 300 μM IPTG and 200 ng/mL aTc were added to the reactions to induce expression. Reactions were set up in Corning CLS3820 plates, with the temperature set to 30°C. Fluorescence measurements were made at an interval of 7 minutes in a Biotek Synergy 2 plate reader. The fluorescence units shown in Figure 4 represent values obtained after 6 hours. In Figure 4 purified TetR was added to a final concentration of 1 μM. Concentrations of DNA and TetR used were based on previous optimizations [24]. The fluorescence values represent an average of three measurements and the error bars indicate the standard deviation.

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Figure 4. Effect of tetO on LacI mediated repression of T7lacO when tetO is in between the two lac operators in cell free systems.

A) Fluorescence response from pDRT7 77 to LacI and TetR proteins. B) Shows fluorescence response from pDRT7 14 and pDRT7 77 plasmids to presence of 300 μM IPTG and/or 200ng/ml aTc. Error bars in the figure depict standard deviations of triplicate measurements.

https://doi.org/10.1371/journal.pone.0078442.g004

Results

Discussion

The field of synthetic biology has seen tremendous progress in the past few years and has led to the development of increasingly sophisticated gene networks. Several examples of transcriptional logic gates have been described using bacterial promoters, and transcriptional elements have been extensively used for construction of small circuits such as logic gates, oscillators and bistable switches. Although engineered control of gene expression is possible with bacterial promoters, implementation of orthogonal networks in bacterial systems is complicated by the shared use of the host’s machinery [35,36]. In contrast to bacterial promoters, however, the use of viral promoters offers a promising path to orthogonality if similar logic control systems can be established. Accordingly, T7 promoters have been used to construct a modular AND gate and to construct orthogonal feed forward loops in E. coli [14,22]. More recently, Shis and Bennet designed an AND gate using a split two component T7 RNA polymerase as an input [21].

However, the number of transcriptional logic control systems utilizing the T7 promoter is limited when compared to those described using bacterial promoters, which take advantage of the concerted action of multiple transcription factors. For instance, Isalan et.al described a T7 promoter regulated by two zinc finger proteins, which was constructed by placing two different operators that bind two zinc finger proteins[28]. Typical bacterial control elements bind cis-regulatory regions that can span more than 100 bp upstream and downstream to the transcription start site [37,38]. Consequently, a broad array of regulation mechanisms can be incorporated that operate either individually or in concert to create programmable, multiple input responsive promoters. As an illustration of the power of this approach, Hunziker and colleagues designed 12 different types of logic functions using a combination of cAMP-CRP activator protein, GalR repressor protein and promoters of diverse strengths [39]. However, the short cis-regulatory region around T7 promoters presents a challenge for implementing logic control using the aforementioned strategies.

In contrast to cis-regulatory strategies, protein dimerization mediated DNA looping permits transcriptional control by proteins bound at locations distal to the promoter in both natural and synthetic promoters. In particular, the role of DNA looping for transcriptional regulation of bacterial promoters by lac[29,33], araBAD[40], gal[41] and cI[42] has been extensively studied in prokaryotes[43]. Additionally, in native E. coli lac promoter systems, looping facilitates tight repression of E. coli lac promoters even at low LacI repressor concentrations[44]. DNA looping has also been employed for constructing logic gates. Zhan et al. utilized two different lacO binding sites with E. coli promoters to introduce looping, thereby enabling regulation by the concerted action of two different repressors bound at different sites[45].

Here, we implemented an IMPLIES gate using a strategy that harnesses DNA looping. While the IMPLIES gate is not commonly used in electrical engineering, the value of constructing an IMPLIES gate described here, lies in the fact that, IMPLIES gates together with NOT gates can be utilized to realize any logic function, as described in Figure S6. Therefore, the IMPLIES gate constructed here provides a powerful way forward for harnessing the orthogonality of T7 promoters to perform any desired logic operation. Here we addressed the challenging aspect of engineering dual control of T7 promoters.

First, we assessed the potential advantages of DNA looping for control of the T7 promoter system. Without looping, very weak repression is observed, as shown by pREPT7 01 in Figure 2B. This may come as a surprise in light of the fact that strong overall induction of gene expression is achieved with the pET system [23]. However, in the pET system, IPTG simultaneously induces both T7 polymerase and the T7LacO promoter, which makes it difficult to assess the true performance of the T7lacO promoter. This is why a strain with arabinose inducible T7 polymerase was chosen. In addition, we induce cells at a low OD value (0.01) as compared to the typical OD values at which the pET system is induced. This can lead to weak accumulation of LacI prior to induction. After characterizing repression without looping, we then inserted LacO1 operator sequences around the T7 promoter. Specifically, a LacO1 auxiliary operator was placed 92 bases upstream from a primary LacO1 operator that resides immediately downstream of the T7 promoter. This architecture enabled strong repression of the T7 promoter in the presence of LacI. As shown in Figure 2B, introduction of an auxiliary LacI binding site resulted in ~3.5-fold increase in LacI mediated repression in comparison to T7lacO promoters lacking the auxiliary distal operator. Additionally, changing the auxiliary operator sequence such that LacI binding was weakened allowed tuning of repression levels from T7lacO promoters. These results mirror the effect of auxiliary operators on expression from E. coli lac promoters[29] and demonstrate the ability to further regulate expression from T7 promoters.

A previous investigation of DNA looping for LacI mediated control of the T7 promoter placed the auxiliary operator 238 bp away from T7lacO promoter, which resulted in only modest repression [23]. The enhanced repression levels reported here likely result from the use of a shorter distance between lac operators[44]. In studies based on E. coli lac promoters, a substantial decrease in repression levels accompanied an increase in operator distance [33]. A 50-fold change in gene expression was observed at an inter-operator distance of 70.5, while only a 15-fold change in expression was found when the distance was 150 bp. At shorter distances, the energy required for formation of a LacI dependent loop between operators located on the same side of the DNA helix is lower than the energy required for formation of a loop between operators that are out of phase[30,44]. Therefore, for the constructs pDRT7 77 and pDRT7 70 (described in Table 1 and Table S1) the inter-operator distances of 92 and 70 were chosen based on in vivo data for LacI mediated transcriptional control with E. coli promoters. The dual binding site design leads to a significant increase in repression levels when compared to a single LacI binding site. In our T7 system, as observed with E. coli promoters, controlling the distance between protein binding sites may enable further tuning of T7lacO promoters (Figure S5).

Having established the value of placing additional lac operator site upstream to T7lacO promoter to regulate transcription, we next sought to place a second regulatory site that might interfere with the looping process thereby interfering with lacI mediated repression. Accordingly, a tet operator was placed 59 bases upstream from the primary lac operator. This positioning of tetO resulted in a 2 to 3 fold change in gene expression upon the addition of aTc and in the absence of IPTG (Figure 3B-C). In contrast, inserting tetO within the LacO looping framework, at -21, -23 and -25 positions resulted in little to no observable reduction in expression in the presence of aTc and in the absence of IPTG when compared to the uninduced state (Figure S4). These results suggest that while TetR bound at tetO centered at the -59 position is optimally placed to interfere with LacI mediated repression of the T7lacO promoter by interfering with the formation of the LacI mediated DNA loop and therefore with LacI mediated repression.

The differences in the response of pDRT7 77, pDRT7 21, pDRT7 23, pDRT7 25 and pDRT7 27 (Table S1) to aTc (Figure 3B and Figure S3) may be explained by the orientation of tetO binding site relative to the DNA loop formed between the two lac operators. The ability of TetR to sterically hinder the formation of the LacI based loop may depend on position. In the case of E. coli promoters, a periodic dependence on distance between the lac operators has been observed [29,33]. A repression maxima is seen for distances that place the operators in phase and occurs with a periodicity of 11 to 11.3 bp with E. coli promoters of 59 bases, 70.5, 81.5 and 92 bases. Minima appear when the operators are placed on the opposite sides of DNA [30,33]. Based on this model, tetO centered 59 bp upstream to the primary lac operator would be in phase with the primary lac operator and may sterically hinder LacI mediated loop formation. Alternatively, TetR interference with LacI mediated repression could result from stiffening of the DNA template by TetR thereby increasing the persistence length of the DNA and making loop formation energetically unfavorable. Although confirmation of the underlying mechanism awaits further experiments, TetR binding to tetO at a distal site interferes with LacI dependent repression and enables an IMPLIES logic function. Moreover, we observe sharp transitions between on and off states. As shown in Figure 2, the Hill coefficient of response to IPTG for pREPT7 11 construct is ~2. Participation of protein tetramers for achieving repression likely contributes to the steepness of the response to IPTG. The result is IMPLIES logic behavior characterized by a sharp transition between the on and the off states.

We caution that, to fully capitalize on the orthogonality offered by T7 transcription, other system components must be chosen carefully with regards to the host genotype. Although we used well-characterized Lac system components that are derived from E. coli, host strains could be chosen to avoid potential interactions. Moreover, cell-free systems can be used to provide a minimal context with reduced chances for host interactions.

The T7 promoter regulated by both LacI and TetR was further tested in cell extracts and constitutes a demonstration of digital logic in a cell-free system using T7 promoters. To test the logic gate, purified TetR was added to the extract, which already contained Lac repressor protein. As with the live cell systems, the presence of TetR protein interferes with LacI based repression, and an IMPLIES logic function is realized (Figure 4B).

Cell-free systems are ideal platforms for developing and implementing simple regulatory circuits. In contrast to the use of live cells, cell-free gene circuits offer the advantage of harnessing biological function without regard for cell survival. To optimize a gene circuit function, different components of the circuit such as the DNA sequence and the transcriptional regulator concentrations can be precisely defined to identify conditions for eliciting the desired response from a gene circuit. In contrast, in live cells these parameters can only be coarsely determined by modulating either the origin of replication to tune DNA copy number or other determinants of genetic expression for tuning regulator concentrations. Consequently, cell-free systems have tremendous potential for implementing predictable and precisely defined gene circuits, as they combine the simplicity of an in vitro system along with the remarkable capability for continuous protein production from DNA encoded instructions[24,46]. Further, control of biochemical components can be combined with defined physical platforms that mimic the size and scale of biological reactions[47]. Consequently, gene circuits and biosynthetic capabilities can be harnessed to evaluate gene circuits and biochemical reactions in cell-free environments [48-52]. Together, our demonstration of dual input regulation of T7 expression and digital logic contributes to a small but growing body of work for construction of more sophisticated cell-free circuits [18,24,53-56].

In future work, several approaches can be taken to optimizing the dynamic range of our components. A more detailed examination of lac operator spacing, as has been done with E. coli promoters, may facilitate tuning of the response to suit downstream application requirements. As described by Khalil et.al, transcriptional output can be tuned through systematic modification of operator count and operator affinity for the transcriptional factor (as shown in Figure 2) [57]. In addition, repressor concentrations may be tuned to optimize dynamic range. However, excessively large induction factors are not always required to construct interested gene circuits. For example, Tabor et al constructed a ‘dark sensor’ optogenetic component with a dynamic range of less than 3-fold yet proceeded to successfully integrate this with other components to construct a bacterial ‘edge detection’ circuit [58]. If necessary, dynamic range can also be expanded through the use of signal amplifying gene circuits, as explained and experimentally demonstrated [59]. Such amplification was realized in the construction of multicellular logic functions by Tamsir et al. [60].

The work described here marks a starting point for using T7 promoters to implement digital logic and other complex functionality in biochemical systems. As described in Figure S6, the IMPLIES gate constructed here may be combined with NOT gates to realize any logic function using T7 transcription. DNA looping harnessed in this strategy not only improves the repression of expression from T7 promoters but expands the otherwise limited region that can be used to exert control over T7 promoters. Overall, the approach put forth here, combined with the existing availability of promoters of varied strength[61], polymerase specificity[20,62] and two component T7 RNA polymerases[21] paves the way towards harnessing the portability of T7 promoters for realizing synthetic networks in cells and in cell-free systems that are more decoupled from cellular processes.

Supporting Information

Acknowledgments

The authors would like to acknowledge Hannah R. Meredith for performing some preliminary experiments that led to this study.