Characterisation of input devices
The choice of input signals presents the first possible complication in terms of parts modularity. For this reason, genetic circuits of higher complexity with multiple inputs often utilise promoter systems which are activated by inducers of vastly dissimilar chemical nature, namely IPTG, tetracycline, arabinose, 3OC12HSL and C4HSL. Previous studies have shown that a subset of quorum sensing promoters can be activated by homoserine lactone inducers of similar carbon chain length [29, 30]. Likewise, the wild-type pBAD promoter is affected by lactose analogues, requiring further mutagenesis to avoid crosstalk inhibition [31]. Instances of cross-phosphorylation have also been observed in two component signal transduction systems between otherwise distinct pathways [32]. Thus, it is important for inducible input devices to be carefully characterised for their steady state transfer function and pairing compatibility before further assembly into higher ordered logic devices.
While previous studies with pRHAB promoter involved genetic circuits that include both RhaR and RhaS transcription factors [33–35], in this paper we demonstrate that the rhamnose inducible promoter pRHAB requires only RhaS for full activation and displays tight regulation even when RhaS is overexpressed. Additional file 1: Figures S2C and S3C show the steady state transfer functions of input device A, pBAD (Additional file 1: Figure S2A) and input device B, pRHAB (Additional file 1: Figure S3A) expressing RFP under strong ribosome binding sites (RBSs) by their corresponding inducers, respectively.
To examine the possibility of genetic cross-communication, we constructed genetic circuits that couple GFP production to pBAD activation and RFP production to pRHAB activation. The results show that varying the concentration of arabinose did not activate pRHAB promoter activity (Additional file 1: Figure S4A). A similar trend was observed in pBAD promoter with rhamnose (Additional file 1: Figure S4B). Interestingly, the simultaneous introduction of both sugars modified the transfer function of each promoter slightly, which may be a result of differential cell growth, sugar import rate or antagonistic effect of one sugar to another. This effect, however, is insignificant as definite ON and OFF switch behaviours are apparent — thereby confirming the pairing compatibility of pBAD and pRHAB promoters.
Design and characterisation of AND logic gate
Designs of highly modularised, prokaryotic AND logic devices have hitherto involved the use of multiple plasmids [10, 12, 16, 36, 37]. In this work, we assembled the AND logic gate in a single plasmid. This procedure has enabled us to localise the AND logic gate in a single vector, and facilitated the downstream troubleshooting and tuning of layered genetic circuits.
To develop the AND logic component of the half adder, we systematically designed and assembled refactored modules of the HrpRS transcription machinery into a low copy plasmid (Additional file 1: Figure S2A). The module which expressed GFP from the pHrpL promoter was assembled upstream of the pBAD-HrpS and pRHAB-HrpR modules to attenuate genetic context-dependent effects that might arise from transcriptional overrun of the stronger pBAD and pRHAB input expression modules as a result of inefficient transcription termination. While designing the GFP producing module in a bidirectional permutation is usually a better solution, this option was not tested in our study, as the downstream pBAD promoter is a weak constitutive promoter in the reverse complement direction. Thus, placing the pHrpL-GFP module before pBAD in either the reverse or reverse complement arrangement may result in antisense-GFP interference or the occurrence of a leaky AND gate. The steady state profile of the functional AND gate was characterised by titrating with a varying concentration of arabinose (input A) and rhamnose (input B) as shown in Fig. 2b. Results of the engineered AND gate correlated well with our steady state computational model (Additional file 1: Figure S11), which was applied to match biological modules making up the AND gate. Likewise, the ‘on’ and ‘off’ digital performance of the AND gate at steady state was qualitatively and quantitatively assessed by introducing inputs well above switch points under four different logic conditions (Fig. 2a and c). The results show that the AND gate was only activated in the presence of both inputs with >800au (relative fluorescence unit) expression increase, as compared to the condition where only a single input is present (or no inputs).
To assess the effect of plasmid copy number on the performance of the AND gate, modules were constructed which generate the HrpRS transcription activators (pBAD-HrpS-pRHAB-HrpR). This produces a GFP output (pHrpL-GFP) into separate low and high copy plasmids (co-transforming the plasmids into E. coli cells).The relative GFP output of each system was measured (Fig. 2d). The results show that the AND gate system with the GFP-producing module in the high copy plasmid and the HrpRS transcription activators in the low copy plasmid produced a >4-fold greater GFP output than AND gate systems with GFP-producing module in low copy plasmid and HrpRS (as compared to transcription activators in either low or high copy plasmids). The result indicates that a higher concentration of HrpRS transcription activators, above the saturation limit of the pHrpL promoter, do not produce a greater GFP output. It is likely that the transcriptional output of the HrpRS AND gate is limited by the strength of the weak pHrpL promoter. Hence, the conclusion is that when pHrpL-GFP module was expressed in high copy plasmids, the intracellular availability of pHrpL promoters was increased — resulting in the amplification of GFP output.
Design and characterisation of OR logic gate
Genetic OR gates can be achieved by designing tandem promoter genetic circuits or by expressing target genes in two discrete expression cassettes. Nonetheless, tandem promoter OR gate circuits may fail when repression of the downstream promoter prevents the proper functioning of the upstream promoter [38]. To develop the OR logic gate of the half adder, three prototype designs were constructed; two designs comprised pBAD and pRHAB promoters in different tandem arrangements upstream of an RFP reporter gene with strong RBS, and a third design produced RFP in two distinct expression cassettes (Fig. 3a). The three OR gate designs were then introduced with input A and B above their switch points and assessed for the respective RFP outputs (Fig. 3a and c). The results show that designs I and III are functional OR gates with >2,500 au higher RFP expression when either or both inputs are present. In our computational model, the total amount of RFP expression was approximated by the sum of RFP amounts produced from individual pBAD and pRHAB promoters. Although the model predicts well from low to medium range induction levels, our assumption was not valid at very high induction levels, in which lesser RFP expression was observed than predicted. It is possible that at very high induction levels, the transcription and translation machinery in cells are fully saturated, thereby imposing metabolic burden on the cells and limiting protein production [39]. The OR gate design II, which was composed of the pRHAB promoter upstream of the pBAD promoter and RFP reporter, was activated only in the presence of rhamnose, but not arabinose. Our results agree with the previous finding that no expression was detected when the pBAD promoter was fused downstream of the tetracycline-inducible pTET promoter and upstream of a YFP reporter [38]. We conclude that this observation is likely an effect of the AraC transcription factor, which can function as both repressor and activator. In the absence of arabinose, AraC, when overexpressed, remains bound to operator sites that induce DNA looping of the pBAD promoter, thereby obstructing the elongation of mRNA by initiated RNA polymerase. As will be shown in the next section, in order to layer OR gate design I into other logic devices, the construct was characterised for its steady state profile by titrating with varying concentrations of arabinose and rhamnose (Fig. 3b). Results of the engineered OR gate generally correlated well with our steady state computational model (Additional file 1: Figure S12), which was applied to match biological modules making up the OR gate.
Genetic context effect of σ54-dependent pHrpL promoter
To enable sufficient expression of the λCl repressor by an AND gate system, the gene encoding for λCl repressor was assembled downstream of a σ54-dependent pHrpL promoter on a high copy plasmid. Fortuitously, we discovered that a pHrpL promoter located downstream of another pHrpL expression cassette can be turned on even in the absence of its cognate HrpRS transcription factors (Additional file 1: Figure S5C). The converse is not true for an upstream pHrpL promoter (Additional file 1: Figure S5B). Negative controls with just the GFP reporter or RBS-λCl gene upstream of the pHrpL-GFP module confirmed that the pHrpL promoter alone is not leaky and that a cryptic promoter is absent in the λCl gene (Additional file 1: Figure S5A and S5D). To buffer against this genetic context-dependent effect of the pHrpL promoters, pHrpL-GFP and pHrpL-λCl modules were assembled on separate plasmids. This successfully prevented the genetic interference of both pHrpL expression modules with each other (Additional file 1: Figure S5E and S5F). Additional file 1: Figure S5G shows a quantitative assessment of pHrpL promoter activation due to the presence of another upstream pHrpL promoter and the use of plasmids as genetic insulators.
Design and characterisation of NOT and NIMPLY logic gates
As part of the development of XOR logic operations of the half adder, repressor binding sites are required downstream of the OR gate promoters. To examine the minimal number of λCl repressor binding sites required for effective repression, a single λCl operator site and dual λCl operator sites of perfect dyad symmetry were fused downstream of the pBAD promoter, before the RFP gene [40]. The repressibility of both circuits was tested by generating λCl repressors from an HrpRS AND gate in a separate plasmid. Negligible repression was observed when only one λCl repressor operator site was present. In the presence of two operator sites of perfect dyad symmetry, RFP expression from the pBAD promoter was greatly attenuated — even when the λCl repressor was not synthesised. We postulate that the observed reduction of RFP expression might be caused by the presence of secondary hairpin structures immediately downstream of the TSS acting as pseudo transcription terminators or locking the RBS in conformations that prevented translation initiation (Additional file 1: Figure S6A).
In order to examine this further, random mutagenesis on the natural sequence of the λCl repressor operator sites was performed with screening for mutants with significant difference in RFP expression levels, in the absence and presence of the λCl repressor. Accordingly, an evolved candidate (Cl2B) with 4 mutations in the inverted sequence of the λCl repressor binding (Additional file 1: Figure S6B) was obtained. Sequence comparison with the original λCl repressor binding sites (Cl2A) with the evolved candidate revealed that the directed evolution process had eliminated the effect of secondary hairpin structures from 7 to 3. Next, the efficiency of λCl-mediated transcription termination in the context of a genetic NIMPLY gate was studied. This was achieved by placing repressor binding sites directly downstream of tandem pBAD-pRHAB promoters and generating λCl repressors from a separate pBAD expression cassette.
Two NIMPLY logic circuits were developed which generated RFP transcripts with strong and weak RBSs. Both NIMPLY logic circuits were then tested in the presence and absence of input A (arabinose) over time with input B (rhamnose), both above the switch point (Fig. 4a). Temporal analysis of the NIMPLY logic circuits showed that there was no significant delay in layering a NOT gate downstream of an OR gate (Fig. 4b). However, an apparent delay in the total amount of mature RFP was observed when a weaker RBS was used. The results also showed that while NIMPLY logic can be achieved from both circuits, the system with the strong RBS exhibited a higher order of expression and leakiness compared to that which translated RFP from the weaker RBS. This leads to the conclusion that the choice of a particular RBS can be used as a signal moderation technique in order to achieve a balance between precision tuning and output gain in layered logic gates. In an attempt to alleviate expression leakiness from the NIMPLY gate with the strong RBS, an additional pair of λCl repressor binding sites with imperfect dyad symmetry were introduced downstream of pBAD-pRHAB-Cl2B, and before the RBS-RFP module. However, the presence of 4 λCl binding sites completely inhibited RFP expression, resulting in the failure of the NIMPLY gate (Fig. 4c). It is likely that this failure could be an effect of pronounced 5′ UTR secondary structures formed due to the repeated use of identical λCl repressor binding sites.
Design and characterisation of XOR logic gate
In order to develop the XOR component of the half adder, we assimilated and tested a combination of AND, OR and NOT logic gates in four different genetic circuits. In all the designs HrpRS transcription activators were expressed from low copy plasmids to drive the synthesis of λCl repressors from the pHrpL promoter in high copy plasmids (Fig. 5b). OR and NOT biological modules were assembled in the same high copy plasmid downstream of the pHrpL-λCl module. In design I, an OR gate comprising a tandem arrangement of pBAD, pRHAB and λCl repressor binding sites was used to express ssrA-tagged, short-lived RFP (RFPasv) — one of the most well-characterised protein degradation systems in E. coli [41]. In design II we created hybrid promoters of pBAD and pRHAB by incorporating λCl binding sites downstream of both promoters before connecting them in tandem to elicit hypothetical OR logic similar to design I. Design III was modified from design II to express long-lived RFP. To overcome possible complications from 5′UTR secondary structures — due to the presence of multiple λCl binding sites within the same mRNA transcript — design IV, which comprised synthetic hybrid promoters of pBAD-Cl2B and pRHAB-Cl2B expressing RFPasv in two discrete expression cassettes, was also developed.
Accordingly, only design IV was able to achieve well-balanced outputs which accurately described XOR logic operations (Fig. 5c). While design I demonstrated the strong suppression of RFP output in the presence of both inputs (arabinose and rhamnose), when characterised as a NIMPLY gate (as described earlier), the same design failed to function in the context of a XOR gate in which a weaker pHrpL promoter was used to drive the synthesis λCl repressors instead of the strong pBAD promoter. Interestingly, the results imply that when employing transcription repressors as molecular blockers to mRNA elongation, a higher concentration of λCl molecules is needed to completely suppress transcription as λCl binding sites are engineered further away from the transcription start site. This observation may be an effect of RNAP gaining momentum as it runs down template DNA to perform transcription, inadvertently enabling RNAP to continue its course of action as a result of the inadequacy of ‘molecular brakes’.
While designs II and III, which were developed with λCl binding sites downstream of both pBAD and pRHAB promoters, exhibited a slight semblance of XOR logic operations, the presence of multiple, repeated sequences of λCl binding sites in the transcript generated from the pBAD promoter greatly reduced the RFP output from input A. Using untagged RFP gene in design III led to a slight increase in overall RFP output but did not alleviate the signal balancing issue. The result implies that the 5′UTR structural effect is more dominant than RFP half-life in determining the success of the layered XOR gate. In order to apply the XOR gate in the implementation of the half adder, design IV was characterised for its steady state profile by titrating with varying concentrations of arabinose and rhamnose as shown in Fig. 5d. It is noteworthy that the XOR gate developed in this work possesses higher single cell computational capability compared to that achieved by Tamsir and colleagues using a network of inter-communicating cells [38], hence circumventing problems associated with cell-cell communication.
Design and characterisation of single cell half adder and half subtractor
The half adder computes dual inputs with both AND and XOR logic operations to generate CARRY and SUM outputs, respectively. Building on bio-logical devices that were modularised and rigorously characterised earlier, we co-transformed constructs which produce GFP (CARRY) from an HrpRS AND gate in a low copy plasmid, RFPasv (SUM) from hybrid promoters pBAD-Cl2B and pRHAB-Cl2B and λCl repressors from a pHrpL promoter in a high copy plasmid into E. coli (Fig. 6a). To study the digital performance of the single cell half adder, we characterised the system at both the population and single cell levels by microplate fluorescent assay (Fig. 6b) and flow cytometry (Fig. 6c, Additional file 1: Figure S7) for four different logic conditions. The results show that the engineered cells exhibited robust and digital-like performance with minor expression leak (<20 %) in XOR output when both inputs were present. While previous characterisation with standalone XOR gates displayed near perfect XOR outputs, parallel implementation of both AND and XOR logic gates in a half adder led to probable competition for HrpRS transcription activators by pHrpL promoters in both low and high copy plasmids, which is suggestive of expression shunting in competitive transcription dynamics [42]. In other words, the availability of HrpRS activators is divided between the pHrpL-GFP module in low copy plasmids and pHrpL-λCl module in high copy plasmids, thus causing both AND and XOR gates to perform below par compared to when they are operating individually. To affirm the hypothesis, we examined the AND output of a standalone AND gate with the AND output of the half adder using microplate fluorescent assay. The results showed that the GFP output of the isolated AND gate was approximately 7 times stronger than that of the half adder’s AND gate, thus confirming our hypothesis (Additional file 1: Figure S8). It is noteworthy that the reduced expression of GFP did not affect the overall performance of the half adder, as effective half adder logic operations were still achieved. In the current single cell half adder, the engineered cells exhibited relatively healthy growth with the same order of viable cells (about 109 cfu/ml) in both induced and uninduced cell cultures (Additional file 1: Figure S9). Nevertheless, as genetic complexity and heterologous expression increased, a concomitant increase in the metabolic burden in the E. coli cell was also observed.
To demonstrate the modularity of our approach, we also developed a single cell half subtractor by performing slight modifications to the genetic circuits that formed the basis of the half adder. Specifically, GFP, which exemplifies BORROW output, was produced from the hybrid promoter pBAD-Cl2B in the low copy plasmid instead of the pHrpL promoter (Fig. 7a). As above, the construct which generated the BORROW output (GFP) and that which generated the DIFFERENCE output (RFP) were co-transformed into E. coli cells. Characterisation was undertaken at both the population and single cell levels by microplate fluorescent assay (Fig. 7b) and flow cytometry (Fig. 7c) under four different logic conditions. The results showed that the engineered cells functioned as effective biological half subtractors, producing GFP only in the presence of input A and RFP in the presence of input A or B, but not when both inputs were present.