BACKGROUND
gene regulation in biological systems behaves like a molecular computer whereby the gene’s output can be modelled as on-off states of boolean  logic .
however, programming gene regulation is far from trivial and requires considerable time and effort during functional testing and tuning of the synthetic genetic circuits under development.
apart from the scarcity of reliable and well-characterised biological parts, digital performance in biological systems is further impacted by the cellular and genetic context-dependent effects of the biological parts which comprise the circuit .
recent studies have shown that genetic crosstalk between the engineered circuits and endogenous networks of the host cell can lead to cellular context-dependent effects  <cit> .
for this reason, molecular parts and devices that are orthogonal to the cell native machineries with roles in either genetic transcription or protein translation have been created to enable predictable engineering of genetic circuits .
demonstrations of layered genetic circuits in a single cell, such as the execution of a 4-input and gate in bacteria  <cit>  and biological half adders and half subtractors in mammalian cells  <cit>  have revealed that orthogonal logic gates can be interlinked to perform digital operations of higher complexity and diversified outputs.
while the capability to program cells with memory and decision-making functions  presents many opportunities in biotechnological applications, a lack of formal understanding associated with genetic context-dependent effects has limited progress in engineering biology.
in this respect, two studies have shown that the 5′ untranslated region  of mrna can affect the temporal control of multigene operons or inverter-based genetic circuits, and rna processing using clustered regularly interspaced short palindromic repeats  or ribozymes can serve as effective genetic insulators to buffer such context-dependent effects  <cit> .
in this paper, we have sought to elucidate the limitations of engineering biology from an architectural point of view, with the aim of creating a set of engineering solutions for overcoming failure modes during the development of complex, synthetic genetic circuits.
design of biological half adder
in this study we were interested in developing biological half adders in prokaryotic systems — particularly in microbes which exhibit much faster cell division and shorter cycle time — so that they can be broadly applied in different biotechnological applications.
in contrast to the mammalian cell-based half adder, which is developed mainly for therapeutic and biosensing applications, a prokaryotic half adder can be used to enhance molecular process control and decision making, for example, in drug and biofuel production, biosensing, bioremediation  <cit>  and probiotic engineering for the treatment of metabolic disorders  <cit> , cancer  <cit>  and infectious diseases  <cit> .
in digital processing, half adders form the key building blocks for shift registers, binary counters and serial parallel data converters.
likewise in biological systems, a combination of half adders can be connected in various arrangements to regulate gene expression with diverse, digital-like performance.
in doing so, biological systems can be made to interface with novel biomolecular devices, allowing the repurposing of cellular phenotype, as well as providing new platforms to probe and elucidate biological functions .
escherichia coli was chosen as the designated chassis as it represents a model organism that can be easily manipulated; its inherent cellular processes are also well characterised.
figure  <dig> shows the design of our biological half adder in a single prokaryotic cell.
the half adder consists of three independent biologically derived and, or and not logic gates and a fourth and logic function that is not a physical device, but a result of programmable decision making as a result of interconnecting logic functions .
the σ54-dependent hrprs regulation motif of the pseudomonas syringae t3ss secretion system was refactored for the design of the and gate, as demonstrated in an earlier study  <cit> .
the advantage is that the hrprs and gate offers a dual layer of orthogonal control in the e. coli host.
this means that  the majority of transcription events in e. coli will occur via σ70-dependent transcription, and  hrprs transcription factors are absent in wild type e. coli.
transcription occurs when enhancer-binding proteins hrps and hrpr, which are regulated by arabinose  and rhamnose  induction, respectively, are coexpressed and bound to the upstream activator sites of the phrpl promoter.
this binding event then triggers an atpase-dependent conformational change within the promoter through a molecular interplay with the σ54-rnap holoenyzme, thereby allowing rna synthesis and elongation after the transcription start site.
the or gate generates mrna transcript of the rfp gene upon induction with either arabinose or rhamnose.
the not gate in the half adder design is a hybrid promoter consisting of λcl repressor binding sites downstream of the transcriptional start site  of the or logic gate.
unlike traditional not gates, which are designed to have transcriptional repressors competing for consensus rnap binding sites, our not gate design functions as an orthogonal, molecular blocker to the rna elongation process.fig.
<dig> simplified schematics of the biological half adder, comprising independent modules of the and, or and not gates layered in series and in parallel.
a logic output of biological half adder.
b in the presence of two inputs, the and gate is activated to produce gfp and lambda repressors, which further inactivates the or gate to suppress rfp expression.
c in the presence of either inputs singly, only the or gate is activated to trigger rfp expression
on induction with arabinose and rhamnose, the transcription factors arac and rhas, both of which are constitutively expressed in a single transcript by promoter pcon, associate with their corresponding inducers to activate expression of the enhancer-binding proteins hrps and hrpr.
this results in the activation of the and logic and the concurrent synthesis of gfp reporter and lambda repressor  by the phrpl promoter.
consequently, genetic events of the or gate, which run in parallel with the hrprs and gate, are then turned off due to obstructive repression by λcl molecules.
in all, the half adder demonstrates both and  and xor  logic operations; the latter operation is a processed outcome achieved by sequential and parallel layering of and, or and not logic .
by comparison, induction with either inducer singly will trigger only genetic operation of the or gate, resulting in the synthesis of rfp reporter, but not gfp and λcl molecules .
finally, we also demonstrate the development of a single cell prokaryotic, half subtractor via slight modifications to the half adder circuit.
RESULTS
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  <cit> .
likewise, the wild-type pbad promoter is affected by lactose analogues, requiring further mutagenesis to avoid crosstalk inhibition  <cit> .
instances of cross-phosphorylation have also been observed in two component signal transduction systems between otherwise distinct pathways  <cit> .
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 , 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  and input device b, prhab  expressing rfp under strong ribosome binding sites  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 .
a similar trend was observed in pbad promoter with rhamnose .
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  <cit> .
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 .
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  and rhamnose  as shown in fig.
2b.
results of the engineered and gate correlated well with our steady state computational model , 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 .
the results show that the and gate was only activated in the presence of both inputs with >800au  expression increase, as compared to the condition where only a single input is present .fig.
<dig> design and characterisation of the biological and gate.
a design and logic output of hrp-based and gate.
the and gate comprises hrps and hrpr transcription factors that are unregulated under the control of pbad and prhab promoters, respectively.
in the presence of both inputs hrprs jointly bind and induce conformational change in the phrpl promoter, thereby enabling dna transcription and the expression of gfp reporter.
b steady state profile of the and gate for various concentrations of arabinose  and rhamnose .
c digital performance of and gate at steady state.
d characterisation of the hrp-based and gate in both high and low copy plasmids.
the input devices generating hrprs transcription factors and phrl-gfp reporter module are placed in plasmids of different copy numbers to study the effect of plasmid copy on precision control and tuning of hrp-based and gate.
error bars represent the standard deviation of three independent experiments
to assess the effect of plasmid copy number on the performance of the and gate, modules were constructed which generate the hrprs transcription activators .
this produces a gfp output  into separate low and high copy plasmids .the relative gfp output of each system was measured .
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 .
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  <cit> .
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 .
the three or gate designs were then introduced with input a and b above their switch points and assessed for the respective rfp outputs .
the results show that designs i and iii are functional or gates with > <dig>  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  <cit> .
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  <cit> .
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 .
results of the engineered or gate generally correlated well with our steady state computational model , which was applied to match biological modules making up the or gate.fig.
<dig> design and characterisation of biological or gates.
a the genetic blueprint and logic output of three or gate designs.
designs i and ii are tandem promoters in opposite arrangement, while design iii expresses rfp reporter in two distinct transcripts.
only design i and iii are functional or gates that generate rfp in the presence of either inputs.
b steady state profile of or gate i for various concentrations of arabinose  and rhamnose .
c digital performance of or gates at steady state.
error bars represent the standard deviation of three independent experiments
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 .
the converse is not true for an upstream phrpl promoter .
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 .
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 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  <cit> .
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 .
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  with  <dig> mutations in the inverted sequence of the λcl repressor binding  was obtained.
sequence comparison with the original λcl repressor binding sites  with the evolved candidate revealed that the directed evolution process had eliminated the effect of secondary hairpin structures from  <dig> to  <dig>  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  over time with input b , both above the switch point .
temporal analysis of the nimply logic circuits showed that there was no significant delay in layering a not gate downstream of an or gate .
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  <dig> λcl binding sites completely inhibited rfp expression, resulting in the failure of the nimply gate .
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.fig.
<dig> design and characterisation of the biological nimply gate.
a genetic blueprint and logic output of nimply gate.
the nimply gate is designed by incorporating synthetic lambda repressor binding sites downstream of or gate promoters and regulating the expression of lambda repressors through the pbad promoter.
rfp is expressed only in the presence of input b, rhamnose.
b characterisation of nimply gate with different ribosome binding sites.
at steady state nimply gate which utilizes a weaker ribosome binding site  directly upstream of the rfp reporter  exhibits better control and reduced expression leak, as compared to the nimply gate design that contains a stronger ribosome binding site .
expression leakiness in circuits with strong and weak ribosome binding sites after  <dig> hours are denoted by green and orange arrowheads, respectively.
constructs that were singly induced with input b, induced with both inputs a and b, and uninduced are represented by r, a + r and nc as shown.
c characterisation of nimply gates with two  and four  lambda repressor binding sites.
the black line represents empirically derived transfer function for the construct with dual lambda repressor binding sites, as described by the equation provided.
constructs were induced with a fixed amount of rhamnose  and titrated with various concentrations of arabinose .
an increased number of repressor binding sites disrupted the nimply gate, possibly due to pronounced effect of 5′ mrna secondary structures.
error bars represent standard deviation of three independent experiments
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 .
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  — one of the most well-characterised protein degradation systems in e. coli  <cit> .
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.fig.
<dig> design and characterisation of biological xor gates.
a the logic output of xor gate.
b genetic blueprint of four biological xor gate designs.
the xor gate comprises serially layered and, not and or gates.
hrprs transcription factors are carried in a low copy plasmid, while phrpl-λcl and distinct modules of or gates with lambda repressor binding sites expressing rfp reporter are carried in high copy plasmids.
design i comprises tandem promoters with repressor binding sites downstream of prhab promoter and an rfp reporter engineered with the asv protein degradation tag.
designs ii and iii comprise tandem promoters with repressor binding sites downstream of each promoter and rfp with and without the asv degradation tag, respectively.
design iv is modified from design ii with rfp expressed in two disparate transcripts.
c digital performance of various designs of biological xor gates at steady state.
d the steady state profile of xor gate iv for various concentrations of arabinose  and rhamnose .
error bars represent the standard deviation of four independent experiments
accordingly, only design iv was able to achieve well-balanced outputs which accurately described xor logic operations .
while design i demonstrated the strong suppression of rfp output in the presence of both inputs , when characterised as a nimply gate , 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  <cit> , 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  from an hrprs and gate in a low copy plasmid, rfpasv  from hybrid promoters pbad-cl2b and prhab-cl2b and λcl repressors from a phrpl promoter in a high copy plasmid into e. coli .
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  and flow cytometry  for four different logic conditions.
the results show that the engineered cells exhibited robust and digital-like performance with minor expression leak  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  <cit> .
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  <dig> times stronger than that of the half adder’s and gate, thus confirming our hypothesis .
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  in both induced and uninduced cell cultures .
nevertheless, as genetic complexity and heterologous expression increased, a concomitant increase in the metabolic burden in the e. coli cell was also observed.fig.
<dig> design and characterisation of the biological half adder.
a genetic blueprint of the half adder.
b digital performance of the half adder at steady state.
c flow cytometry analysis of the half adder.
the y axis coordinate represents population count, while fitc-a and pe-cf594-a represent channels that detect gfp and rfp fluorescence, respectively.
population shifts to the right represent on behaviour.
error bars represent the standard deviation of four independent experiments
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 .
as above, the construct which generated the borrow output  and that which generated the difference output  were co-transformed into e. coli cells.
characterisation was undertaken at both the population and single cell levels by microplate fluorescent assay  and flow cytometry  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.fig.
<dig> design and characterisation of the biological half subtractor.
a genetic blueprint of the half subtractor.
b digital performance of half subtractor at steady state.
c flow cytometry analysis of the half adder.
y axis coordinate represents population count, while fitc-a and pe-cf594-a represent channels that detect gfp and rfp fluorescence, respectively.
population shifts to the right represent on behaviour.
error bars represent the standard deviation of four independent experiments
CONCLUSIONS
logic gates are the basis of all electronic digital devices, from mobile phones to microprocessors to computers.
they are therefore the basis of the processing of information and control systems.
similarly, the development of biologically based logic gates and logical devices has major potential in terms of information processing and control.
the design and testing of a half adder, which is the subject of this paper, is seen as a significant step in the development of biological logical devices, comprising multiple gates that work stably and in unison.
immediate areas of application are in advanced biosensors.
in the longer term, there is the potential to develop biologically based devices for information processing and control, for example, in the application of human-imposed intra-cellular control.
the underlying strategy of the paper includes applying systematic design through the application of engineering principles  <cit> .
using a forward engineering approach that is supported by modelling and rigorous characterisation, independent modules that enable programmable digital operations in prokaryotic cells, including simple genetic switches, and and, or and not logic operations were systematically assembled and characterised.
and, or and not logic gates were then layered in both parallel and serial arrangements to generate a repertoire of cellular boolean operations that include nimply, xor, half adder and half subtractor logic operations.
using a bottom-up approach for constructing biological systems of increasing complexity, we assessed genetic architectures that led to genetic context-dependent effects.
on this basis, the significance of each design on the overall digital performance of programmable logic gates in engineered cells was studied, leading to the compilation of a comprehensive set of guidelines for troubleshooting synthetic genetic circuits .
this work together with recent studies conducted elsewhere highlight the importance of modularity and characterisation during the systematic layering of multiple biological devices  <cit> .table  <dig> failure modes and engineering solutions for the design and building of layered genetic circuits in a single  cell
overall, the presence of secondary structures in the 5′-utr of mrna affects genetic expression most.
we discovered that the presence of seven consecutive hairpins immediately downstream of the promoter transcription start site would severely impede gene expression.
although an or gate design made up of tandem promoters can be subjected to the undesirable effects of the 5′-utr secondary structure, we showed that the effect is not pronounced in the digital performance of the or logic when the promoters and dna operator sites involved are of markedly different dna sequences.
the or gate design that comprises a separate gene expression cassette also reliably demonstrates digital operation.
however, the involvement of larger dna modules and repetitive use of transcription terminators that are rich in secondary hairpin structures may impede system assembly in terms of construction efficiency and accuracy.
where identical dna sequences are incorporated in a single mrna transcript, as shown in designs ii and iii of the xor gate, the effect of 5′-utr secondary structure preventing gene expression is significantly more pronounced.
thus, it is proposed that xor gate logic in layered genetic circuits should be designed with two discrete expression cassettes instead of employing a tandem promoter circuit design.
it would also be interesting to test if rna processing tools can be employed in multiplex mode to insulate the myriad of biological devices from rna genetic context-dependent effects in layered genetic circuits concurrently.
perhaps of particular interest, we discovered that σ <dig> promoters can exhibit genetic context-dependent effects if two σ <dig> promoters are placed close to each other.
previously, σ54-dependent ntrc-binding promoters have been reported.
these promoters permit transcription in vitro in the absence of enhancer-binding proteins and atp under conditions that promote dna melting.
these include dna supercoiling, temperature rise and lower ionic strength, or the implemention of characteristic point mutations on the σ <dig> protein  <cit> .
in this paper, we show that an upstream σ <dig> phrpl promoter could also activate a downstream phrpl promoter in vivo if the two promoters are in close proximity — possibly as a result of plasmid dna supercoiling.
this undesired switched-on activity can be avoided by designing phrpl expression modules in different plasmids, that is, using plasmids as genetic buffers to insulate such genetic context-dependent effects.
while recombinases have been intelligently crafted into boolean logic gates with dna-encoded memory functions, it is important to note that biosensors connected in and, or and xor operations with recombinase-based logic gates may not be able to distinguish inputs from different environments and provide the desired response.
for example, a probiotic that is genetically programmed in and logic to sense two inputs such as hypoxia and low ph may be activated for hypoxia and low ph signals in two different locations, as compared to sensing both signals in situ.
the same may be applicable for other logic operations with recombinase-based logic gates.
thus, layered genetic circuits that are capable of sensing and providing location-sensitive boolean logic operations are still useful in programming cellular behaviour.
of particular interest is a combination of layered genetic circuits with the synthesis of recombinases as an intermediary output.
this may provide a novel and better platform for programmable cellular behaviour in terms of both accuracy and memory.
with a few notable exceptions  <cit> , most studies of synthetic biological systems are centred on the development of rational engineering approaches, reporting successful and advantageous aspects of the engineered systems, with a lesser focus on reporting failure modes and compiling the engineering solutions applied to troubleshoot system failures.
as synthetic biology moves forward with greater focus on scaling the complexity of engineered genetic circuits, studies which thoroughly evaluate failure modes and engineering solutions will serve as important references for future design and development of synthetic biological systems.

 gene regulation in biological systems behaves like a molecular computer whereby the gene’s output can be modelled as on-off states of boolean logic. however, programming gene regulation is far from trivial and requires considerable time and effort during functional testing and tuning of the synthetic genetic circuits under development. apart from the scarcity of reliable and well-characterised biological parts, digital performance in biological systems is further impacted by the cellular and genetic context-dependent effects of the biological parts which comprise the circuit. recent studies have shown that genetic crosstalk between the engineered circuits and endogenous networks of the host cell can lead to cellular context-dependent effects. for this reason, molecular parts and devices that are orthogonal to the cell native machineries with roles in either genetic transcription or protein translation have been created to enable predictable engineering of genetic circuits. demonstrations of layered genetic circuits in a single cell, such as the execution of a 4-input and gate in bacteria and biological half adders and half subtractors in mammalian cells have revealed that orthogonal logic gates can be interlinked to perform digital operations of higher complexity and diversified outputs. while the capability to program cells with memory and decision-making functions presents many opportunities in biotechnological applications, a lack of formal understanding associated with genetic context-dependent effects has limited progress in engineering biology. in this respect, two studies have shown that the 5′ untranslated region of mrna can affect the temporal control of multigene operons or inverter-based genetic circuits, and rna processing using clustered regularly interspaced short palindromic repeats or ribozymes can serve as effective genetic insulators to buffer such context-dependent effects. in this paper, we have sought to elucidate the limitations of engineering biology from an architectural point of view, with the aim of creating a set of engineering solutions for overcoming failure modes during the development of complex, synthetic genetic circuits. design of biological half adder in this study we were interested in developing biological half adders in prokaryotic systems — particularly in microbes which exhibit much faster cell division and shorter cycle time — so that they can be broadly applied in different biotechnological applications. in contrast to the mammalian cell-based half adder, which is developed mainly for therapeutic and biosensing applications, a prokaryotic half adder can be used to enhance molecular process control and decision making, for example, in drug and biofuel production, biosensing, bioremediation and probiotic engineering for the treatment of metabolic disorders, cancer and infectious diseases. in digital processing, half adders form the key building blocks for shift registers, binary counters and serial parallel data converters. likewise in biological systems, a combination of half adders can be connected in various arrangements to regulate gene expression with diverse, digital-like performance. in doing so, biological systems can be made to interface with novel biomolecular devices, allowing the repurposing of cellular phenotype, as well as providing new platforms to probe and elucidate biological functions. escherichia coli was chosen as the designated chassis as it represents a model organism that can be easily manipulated; its inherent cellular processes are also well characterised. figure  shows the design of our biological half adder in a single prokaryotic cell. the half adder consists of three independent biologically derived and, or and not logic gates and a fourth and logic function that is not a physical device, but a result of programmable decision making as a result of interconnecting logic functions. the σ54-dependent hrprs regulation motif of the pseudomonas syringae t3ss secretion system was refactored for the design of the and gate, as demonstrated in an earlier study. the advantage is that the hrprs and gate offers a dual layer of orthogonal control in the e. coli host. this means that the majority of transcription events in e. coli will occur via σ70-dependent transcription, and hrprs transcription factors are absent in wild type e. coli. transcription occurs when enhancer-binding proteins hrps and hrpr, which are regulated by arabinose and rhamnose induction, respectively, are coexpressed and bound to the upstream activator sites of the phrpl promoter. this binding event then triggers an atpase-dependent conformational change within the promoter through a molecular interplay with the σ54-rnap holoenyzme, thereby allowing rna synthesis and elongation after the transcription start site. the or gate generates mrna transcript of the rfp gene upon induction with either arabinose or rhamnose. the not gate in the half adder design is a hybrid promoter consisting of λcl repressor binding sites downstream of the transcriptional start site of the or logic gate. unlike traditional not gates, which are designed to have transcriptional repressors competing for consensus rnap binding sites, our not gate design functions as an orthogonal, molecular blocker to the rna elongation process.fig. simplified schematics of the biological half adder, comprising independent modules of the and, or and not gates layered in series and in parallel. a logic output of biological half adder. b in the presence of two inputs, the and gate is activated to produce gfp and lambda repressors, which further inactivates the or gate to suppress rfp expression. c in the presence of either inputs singly, only the or gate is activated to trigger rfp expression on induction with arabinose and rhamnose, the transcription factors arac and rhas, both of which are constitutively expressed in a single transcript by promoter pcon, associate with their corresponding inducers to activate expression of the enhancer-binding proteins hrps and hrpr. this results in the activation of the and logic and the concurrent synthesis of gfp reporter and lambda repressor by the phrpl promoter. consequently, genetic events of the or gate, which run in parallel with the hrprs and gate, are then turned off due to obstructive repression by λcl molecules. in all, the half adder demonstrates both and and xor logic operations; the latter operation is a processed outcome achieved by sequential and parallel layering of and, or and not logic. by comparison, induction with either inducer singly will trigger only genetic operation of the or gate, resulting in the synthesis of rfp reporter, but not gfp and λcl molecules. finally, we also demonstrate the development of a single cell prokaryotic, half subtractor via slight modifications to the half adder circuit.  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. likewise, the wild-type pbad promoter is affected by lactose analogues, requiring further mutagenesis to avoid crosstalk inhibition. instances of cross-phosphorylation have also been observed in two component signal transduction systems between otherwise distinct pathways. 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, 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 and input device b, prhab expressing rfp under strong ribosome binding sites 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. a similar trend was observed in pbad promoter with rhamnose. 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. 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. 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 and rhamnose as shown in fig. 2b. results of the engineered and gate correlated well with our steady state computational model, 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. the results show that the and gate was only activated in the presence of both inputs with >800au expression increase, as compared to the condition where only a single input is present.fig. design and characterisation of the biological and gate. a design and logic output of hrp-based and gate. the and gate comprises hrps and hrpr transcription factors that are unregulated under the control of pbad and prhab promoters, respectively. in the presence of both inputs hrprs jointly bind and induce conformational change in the phrpl promoter, thereby enabling dna transcription and the expression of gfp reporter. b steady state profile of the and gate for various concentrations of arabinose and rhamnose. c digital performance of and gate at steady state. d characterisation of the hrp-based and gate in both high and low copy plasmids. the input devices generating hrprs transcription factors and phrl-gfp reporter module are placed in plasmids of different copy numbers to study the effect of plasmid copy on precision control and tuning of hrp-based and gate. error bars represent the standard deviation of three independent experiments to assess the effect of plasmid copy number on the performance of the and gate, modules were constructed which generate the hrprs transcription activators. this produces a gfp output into separate low and high copy plasmids.the relative gfp output of each system was measured. 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. 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. 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. the three or gate designs were then introduced with input a and b above their switch points and assessed for the respective rfp outputs. the results show that designs i and iii are functional or gates with > 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. 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. 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. results of the engineered or gate generally correlated well with our steady state computational model, which was applied to match biological modules making up the or gate.fig. design and characterisation of biological or gates. a the genetic blueprint and logic output of three or gate designs. designs i and ii are tandem promoters in opposite arrangement, while design iii expresses rfp reporter in two distinct transcripts. only design i and iii are functional or gates that generate rfp in the presence of either inputs. b steady state profile of or gate i for various concentrations of arabinose and rhamnose. c digital performance of or gates at steady state. error bars represent the standard deviation of three independent experiments 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. the converse is not true for an upstream phrpl promoter. 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. 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 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. 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. 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 with mutations in the inverted sequence of the λcl repressor binding was obtained. sequence comparison with the original λcl repressor binding sites with the evolved candidate revealed that the directed evolution process had eliminated the effect of secondary hairpin structures from to 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 over time with input b, both above the switch point. temporal analysis of the nimply logic circuits showed that there was no significant delay in layering a not gate downstream of an or gate. 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 λcl binding sites completely inhibited rfp expression, resulting in the failure of the nimply gate. 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.fig. design and characterisation of the biological nimply gate. a genetic blueprint and logic output of nimply gate. the nimply gate is designed by incorporating synthetic lambda repressor binding sites downstream of or gate promoters and regulating the expression of lambda repressors through the pbad promoter. rfp is expressed only in the presence of input b, rhamnose. b characterisation of nimply gate with different ribosome binding sites. at steady state nimply gate which utilizes a weaker ribosome binding site directly upstream of the rfp reporter exhibits better control and reduced expression leak, as compared to the nimply gate design that contains a stronger ribosome binding site. expression leakiness in circuits with strong and weak ribosome binding sites after hours are denoted by green and orange arrowheads, respectively. constructs that were singly induced with input b, induced with both inputs a and b, and uninduced are represented by r, a + r and nc as shown. c characterisation of nimply gates with two and four lambda repressor binding sites. the black line represents empirically derived transfer function for the construct with dual lambda repressor binding sites, as described by the equation provided. constructs were induced with a fixed amount of rhamnose and titrated with various concentrations of arabinose. an increased number of repressor binding sites disrupted the nimply gate, possibly due to pronounced effect of 5′ mrna secondary structures. error bars represent standard deviation of three independent experiments 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. 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 — one of the most well-characterised protein degradation systems in e. coli. 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.fig. design and characterisation of biological xor gates. a the logic output of xor gate. b genetic blueprint of four biological xor gate designs. the xor gate comprises serially layered and, not and or gates. hrprs transcription factors are carried in a low copy plasmid, while phrpl-λcl and distinct modules of or gates with lambda repressor binding sites expressing rfp reporter are carried in high copy plasmids. design i comprises tandem promoters with repressor binding sites downstream of prhab promoter and an rfp reporter engineered with the asv protein degradation tag. designs ii and iii comprise tandem promoters with repressor binding sites downstream of each promoter and rfp with and without the asv degradation tag, respectively. design iv is modified from design ii with rfp expressed in two disparate transcripts. c digital performance of various designs of biological xor gates at steady state. d the steady state profile of xor gate iv for various concentrations of arabinose and rhamnose. error bars represent the standard deviation of four independent experiments accordingly, only design iv was able to achieve well-balanced outputs which accurately described xor logic operations. while design i demonstrated the strong suppression of rfp output in the presence of both inputs, when characterised as a nimply gate, 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, 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 from an hrprs and gate in a low copy plasmid, rfpasv from hybrid promoters pbad-cl2b and prhab-cl2b and λcl repressors from a phrpl promoter in a high copy plasmid into e. coli. 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 and flow cytometry for four different logic conditions. the results show that the engineered cells exhibited robust and digital-like performance with minor expression leak 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. 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 times stronger than that of the half adder’s and gate, thus confirming our hypothesis. 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 in both induced and uninduced cell cultures. nevertheless, as genetic complexity and heterologous expression increased, a concomitant increase in the metabolic burden in the e. coli cell was also observed.fig. design and characterisation of the biological half adder. a genetic blueprint of the half adder. b digital performance of the half adder at steady state. c flow cytometry analysis of the half adder. the y axis coordinate represents population count, while fitc-a and pe-cf594-a represent channels that detect gfp and rfp fluorescence, respectively. population shifts to the right represent on behaviour. error bars represent the standard deviation of four independent experiments 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. as above, the construct which generated the borrow output and that which generated the difference output were co-transformed into e. coli cells. characterisation was undertaken at both the population and single cell levels by microplate fluorescent assay and flow cytometry 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.fig. design and characterisation of the biological half subtractor. a genetic blueprint of the half subtractor. b digital performance of half subtractor at steady state. c flow cytometry analysis of the half adder. y axis coordinate represents population count, while fitc-a and pe-cf594-a represent channels that detect gfp and rfp fluorescence, respectively. population shifts to the right represent on behaviour. error bars represent the standard deviation of four independent experiments  logic gates are the basis of all electronic digital devices, from mobile phones to microprocessors to computers. they are therefore the basis of the processing of information and control systems. similarly, the development of biologically based logic gates and logical devices has major potential in terms of information processing and control. the design and testing of a half adder, which is the subject of this paper, is seen as a significant step in the development of biological logical devices, comprising multiple gates that work stably and in unison. immediate areas of application are in advanced biosensors. in the longer term, there is the potential to develop biologically based devices for information processing and control, for example, in the application of human-imposed intra-cellular control. the underlying strategy of the paper includes applying systematic design through the application of engineering principles. using a forward engineering approach that is supported by modelling and rigorous characterisation, independent modules that enable programmable digital operations in prokaryotic cells, including simple genetic switches, and and, or and not logic operations were systematically assembled and characterised. and, or and not logic gates were then layered in both parallel and serial arrangements to generate a repertoire of cellular boolean operations that include nimply, xor, half adder and half subtractor logic operations. using a bottom-up approach for constructing biological systems of increasing complexity, we assessed genetic architectures that led to genetic context-dependent effects. on this basis, the significance of each design on the overall digital performance of programmable logic gates in engineered cells was studied, leading to the compilation of a comprehensive set of guidelines for troubleshooting synthetic genetic circuits. this work together with recent studies conducted elsewhere highlight the importance of modularity and characterisation during the systematic layering of multiple biological devices.table failure modes and engineering solutions for the design and building of layered genetic circuits in a single cell overall, the presence of secondary structures in the 5′-utr of mrna affects genetic expression most. we discovered that the presence of seven consecutive hairpins immediately downstream of the promoter transcription start site would severely impede gene expression. although an or gate design made up of tandem promoters can be subjected to the undesirable effects of the 5′-utr secondary structure, we showed that the effect is not pronounced in the digital performance of the or logic when the promoters and dna operator sites involved are of markedly different dna sequences. the or gate design that comprises a separate gene expression cassette also reliably demonstrates digital operation. however, the involvement of larger dna modules and repetitive use of transcription terminators that are rich in secondary hairpin structures may impede system assembly in terms of construction efficiency and accuracy. where identical dna sequences are incorporated in a single mrna transcript, as shown in designs ii and iii of the xor gate, the effect of 5′-utr secondary structure preventing gene expression is significantly more pronounced. thus, it is proposed that xor gate logic in layered genetic circuits should be designed with two discrete expression cassettes instead of employing a tandem promoter circuit design. it would also be interesting to test if rna processing tools can be employed in multiplex mode to insulate the myriad of biological devices from rna genetic context-dependent effects in layered genetic circuits concurrently. perhaps of particular interest, we discovered that σ promoters can exhibit genetic context-dependent effects if two σ promoters are placed close to each other. previously, σ54-dependent ntrc-binding promoters have been reported. these promoters permit transcription in vitro in the absence of enhancer-binding proteins and atp under conditions that promote dna melting. these include dna supercoiling, temperature rise and lower ionic strength, or the implemention of characteristic point mutations on the σ protein. in this paper, we show that an upstream σ phrpl promoter could also activate a downstream phrpl promoter in vivo if the two promoters are in close proximity — possibly as a result of plasmid dna supercoiling. this undesired switched-on activity can be avoided by designing phrpl expression modules in different plasmids, that is, using plasmids as genetic buffers to insulate such genetic context-dependent effects. while recombinases have been intelligently crafted into boolean logic gates with dna-encoded memory functions, it is important to note that biosensors connected in and, or and xor operations with recombinase-based logic gates may not be able to distinguish inputs from different environments and provide the desired response. for example, a probiotic that is genetically programmed in and logic to sense two inputs such as hypoxia and low ph may be activated for hypoxia and low ph signals in two different locations, as compared to sensing both signals in situ. the same may be applicable for other logic operations with recombinase-based logic gates. thus, layered genetic circuits that are capable of sensing and providing location-sensitive boolean logic operations are still useful in programming cellular behaviour. of particular interest is a combination of layered genetic circuits with the synthesis of recombinases as an intermediary output. this may provide a novel and better platform for programmable cellular behaviour in terms of both accuracy and memory. with a few notable exceptions, most studies of synthetic biological systems are centred on the development of rational engineering approaches, reporting successful and advantageous aspects of the engineered systems, with a lesser focus on reporting failure modes and compiling the engineering solutions applied to troubleshoot system failures. as synthetic biology moves forward with greater focus on scaling the complexity of engineered genetic circuits, studies which thoroughly evaluate failure modes and engineering solutions will serve as important references for future design and development of synthetic biological systems. 
as synthetic biology moves forward with greater focus on scaling the complexity of engineered genetic circuits, studies which thoroughly evaluate failure modes and engineering solutions will serve as important references for future design and development of synthetic biological systems. here, we have sought to elucidate the limitations of engineering biology from an architectural point of view, with the aim of compiling a set of engineering solutions for overcoming failure modes during the development of complex, synthetic genetic circuits. using a synthetic biology approach that is supported by computational modelling and rigorous characterisation, and, or and not biological logic gates were layered in both parallel and serial arrangements to generate a repertoire of boolean operations that include nimply, xor, half adder and half subtractor logics in a single cell. in particular, the presence of seven consecutive hairpins immediately downstream of the promoter transcription start site severely impeded gene expression. gene regulation in biological systems is impacted by the cellular and genetic context-dependent effects of the biological parts which comprise the circuit. this work describes a representative case study for the debugging of genetic context-dependent effects through principles elucidated herein, thereby providing a rational design framework to integrate multiple genetic circuits in a single prokaryotic cell. subsequent evaluation of these near-digital biological systems revealed critical design pitfalls that triggered genetic context-dependent effects, including 5′ utr interferences and uncontrolled switch-on behaviour of the supercoiled σ promoter.
 gene regulation in biological systems is impacted by the cellular and genetic context-dependent effects of the biological parts which comprise the circuit. here, we have sought to elucidate the limitations of engineering biology from an architectural point of view, with the aim of compiling a set of engineering solutions for overcoming failure modes during the development of complex, synthetic genetic circuits.  using a synthetic biology approach that is supported by computational modelling and rigorous characterisation, and, or and not biological logic gates were layered in both parallel and serial arrangements to generate a repertoire of boolean operations that include nimply, xor, half adder and half subtractor logics in a single cell. subsequent evaluation of these near-digital biological systems revealed critical design pitfalls that triggered genetic context-dependent effects, including 5′ utr interferences and uncontrolled switch-on behaviour of the supercoiled σ promoter. in particular, the presence of seven consecutive hairpins immediately downstream of the promoter transcription start site severely impeded gene expression.  as synthetic biology moves forward with greater focus on scaling the complexity of engineered genetic circuits, studies which thoroughly evaluate failure modes and engineering solutions will serve as important references for future design and development of synthetic biological systems. this work describes a representative case study for the debugging of genetic context-dependent effects through principles elucidated herein, thereby providing a rational design framework to integrate multiple genetic circuits in a single prokaryotic cell. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2015 