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FEBS Letters 582 (2008) 1237–1244

Minireview Mining logic gates in prokaryotic transcriptional regulation networks

Rafael Silva-Rocha, Vı´ctor de Lorenzo* Centro Nacional de Biotecnologı´a, CSIC, Campus de Cantoblanco, Madrid 28049, Spain

Received 10 January 2008; accepted 28 January 2008

Available online 12 February 2008

Edited by Patrick Aloy and Robert B. Russell

and definition of systems boundaries – rather than vague anal- Abstract Prokaryotic transcriptional networks possess a large number of regulatory modules that formally implement many ogies to cutting and pasting DNA sequences. In this mini-re- of the logic gates that are typical of digital, Boolean circuits. view, we briefly assess what is actually available for Yet, natural regulatory elements appear most often compressed designing genetic circuits, how to upgrade natural modules and exaggeratedly context-dependent for any reliable circuit to meet the requirements of robust engineering, and where to engineering barely comparable to electronic counterparts. To find the pieces that are still missing. Furthermore, we raise overcome this impasse, we argue that designing new functions the questions of connectivity and evolvability of biological with biological parts requires (i) the recognition of logic gates modules as two of the major bottlenecks that hinder the devel- not yet assigned but surely present in the meta-genome, (ii) the opment of synthetic biological circuitry. orthogonalization and disambiguation of natural regulatory modules and (iii) the development of ways to tackle the connec- tivity and the definition of boundaries between minimal biological components. 2. De-constructing naturally-occurring genetic circuits into Ó 2008 Federation of European Biochemical Societies. Pub- usable regulatory elements lished by Elsevier B.V. All rights reserved. The principal actors of the biological input/output functions Keywords: Digital circuit; Logic gate; Catabolic regulator; are the cis-(promoters) and the trans-regulatory elements Metagenome; Orthogonalization (transcriptional regulators). Prokaryotic transcriptional factors (TFs) drive the activity of their cognate promoter(s) in response to one or more environmental stimuli. TFs can generally be activators by enhancing the binding or the activity of the RNA polymerase (RNAP) in the cognate promoters, or 1. Introduction repressors by blocking this binding, or both [4]. Most known prokaryotic activators bind the upstream region of a promoter One of the trademarks of Synthetic Biology is the rational in response to a signal (for example, a substrate of the meta- combination of regulatory modules in artificial circuits for per- bolic pathway regulated by the TF) and enhance the recruit- forming non-natural tasks, including complex binary compu- ment of the RNAP to the site. Alternatively, they may tation operations based on logic gates [1,2]. The basis of promote the escape and further progression of the transcrip- such an endeavour is the implicit adoption of the metaphor tion machinery from the promoter into the transcribed DNA of the cell as a sort of Turing machine. In this way, physico- sequence [5]. In contrast, transcriptional repressors typically chemical environmental signals (the inputs) activate an existing interfere with the binding of RNAP to the 35 and 10 gene expression program (encoded in the DNA), which is ulti- DNA hexamers of bacterial promoters. In this case, environ- mately executed by transcriptional regulators on promoters mental stimuli decrease the affinity of the TF for its binding and then by the downstream protein expression machinery site, thereby allowing the RNAP to access the promoter and [3]. This results, e.g. in changes of the cell metabolism through proceed with transcription [6,7]. One question relevant to cir- the increase or decrease of the production rate of specific pro- cuit design emerges now: why activators and repressors instead teins (the output). Under this conceptual frame, the program of just one mechanism or the other? Sometimes the very same behind any biological function could in principle be de-con- biological function (for instance, the ara systems for arabinose structed into minimal operative units, called by many biologi- consumption) is positively regulated in one bacterium (E. coli, cal parts (see http://parts.mit.edu [1]). Such units can then activated by AraC [8]) and negatively controlled in another (B. ideally be re-assembled following a rational blueprint to per- subtillis, repressed by AraR [9]). There is not an easy answer to form a different program, resulting in altogether new proper- this. It seems that activators generally produce more transcrip- ties and behaviours. In this respect, Synthetic Biology clearly tional output than repressors [10]. It is also likely that positive takes off from what since the late 1970s was called Genetic regulation allows a higher connectivity of the corresponding Engineering, as it brings into Biology robust engineering prin- promoter to physiological co-regulation [11]. ciples such as abstraction, hierarchical design, modularization 2.1. Prokaryotic promoters as Boolean logic gates *Corresponding author. Fax: +34 91 585 45 06. The participation of one or more TFs in the regulation of a E-mail address: [email protected] (V. de Lorenzo). given promoter confers the system the ability of integrating

0014-5793/$32.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.01.060 1238 R. Silva-Rocha, V. de Lorenzo / FEBS Letters 582 (2008) 1237–1244 different input signals in a fashion not unlike those described by the gates of Boolean logic. Such gates perform operations on one or more inputs and produce each time a single logic output. Since the output is also a logic-level value, an output of one logic gate can connect to the input of one or more other logic gates. The logic thereby performed is thus adequate for the functioning of digital circuits. Logic gates are typically implemented electronically using diodes or transistors but, as discussed below, can they also be constructed using inter alia promoters and regulators. An archetypical example in this context is the lac operon of E. coli, where expression of the genes for lactose metabolism is controlled by the lacI repressor and by the cyclic AMP receptor protein (CRP) activator. The LacI repressor binds to the lac promoter (Plac) as a tetramer and inhibits gene expression both through the physical occupa- tion of the RNAP binding site and through the formation of a DNA loop [12]. The binding of the inducer (lactose or IPTG) to LacI triggers a conformational switch in the tetramer that decreases the affinity to the operator sequences and thus allows transcription initiation from the Plac [12,13]. The behaviour of the lac regulatory system has been described to be an interme- diate between AND-gate and OR-gate logic function (see be- low; [14]). Although binary logic circuits are based on functions with just two possible states (0 or 1), existing biological systems typi- cally display continuous values for the input/output functions [15]. In addition, such values are submitted to noise and cell- to-cell stochastic variations due to the nature of the molecular interactions involved [16]. This has important consequences for the construction of artificial genetic circuits based in the naturally occurring transcriptional modules and its applicabil- ity in synthetic networks [17]. For example, an artificial system with oscillatory properties constructed by the combination of the repressor properties of three well characterized TFs (LacI, Fig. 1. Models of logic gates built with prokaryotic regulatory TetR and the k repressor), lost its periodicity after a few modules. The sketches on top of the figure symbolize the various rounds of oscillation [18]. Although promoters destined for actors that control promoter activity: RNA polymerase (RNAP) building artificial circuits should ideally behave as bi-stable disclosed in its various subunits, transcription factors (activators and/ or repressors), binding DNA sites and types of interaction. Each of the switches resembling a digital response, this is not the case in gate models are assembled by combination of TFs binding sites most available instances. Whether or not naturally occurring (operators) and RNAP binding sites (promoters). The overlapping of promoters can be artificially re-designed to achieve perma- one operator with a promoter causes repression while operators placed nently such a binary performance remains an open question, upstream from the promoter causes activation. (a) The amplifier-gate as Darwinian selection may eventually press against such a is represented as a simple activation process. (b) The NOT-gate is equivalent to transcriptional repression. (c) The AND-gate can be conduct. implemented as a TF that depends on an inducer B to activate the promoter. (d) The OR-gate could be a promoter amenable to full 2.2. Simple logic gates shape the bulk of transcriptional activation by two independent TFs. (e) One NAND-gate is generated regulation circuits by a promoter regulated by two cooperative repressors. (f) An ANDN- gate can be created with a promoter activated by a TF and repressed Despite the constraints mentioned above, representing the by another. reactions and interactions involved in gene expression control using circuit diagrams and Boolean logic operators is still an useful abstraction. As the biological reactions adopt somewhat continuous values, the 0/1 states are generally agreed to reflect or amplifier-gate, where the output has the same state that the low/high states for the input status and off/on for output pro- input (i.e., if the input is low the output is off and vice-versa, moter activity. Figs. 1 and 2 summarize the most relevant logic Fig. 1A). For a repressor, the system is represented as a gates that have been either described experimentally or sug- NOT-gate, where the promoter is active (on) only in the gested to occur on the basis of simulations using empirical absence of the repressor (the low state, Fig. 1B). The graphical data. The schemes of Figs. 1 and 2 do not cover all possible difference between these two gates is the presence of an invert- combinations of regulatory modules that can originate the di- ing bubble on the output terminal of the NOT-gate. verse gates shown, but they illustrate each case with a simpli- For the systems where two inputs are computed to generate fied biological example. one output, there are 16 possibilities of Boolean logic gates (2n, The two simplest logic gates that describe biological func- where n = 4 combinations of input states) [19]. However, there tions include one promoter regulated by one activator or by seems to be only eight biologically relevant gates, as analyzed one repressor. In the first case we have the so-called buffer-gate previously [2]. The AND-gate represents a regulatory system R. Silva-Rocha, V. de Lorenzo / FEBS Letters 582 (2008) 1237–1244 1239

cases; see [19,22,23]) strengthens the concept that biological circuits are materialized in a configuration that allows the cells to easily re-program the input/output functions as the result of a simple Darwinian selection process for a specific behaviour.

2.3. Recognition of composite logic gates in complex prokaryotic promoters Apart of the simple gates discussed above, prokaryotic tran- scription networks also provide numerous instances of composite logic operators that are implemented through more complex natural or simulated regulatory setups. For instance the NAND-gate (NOT-AND) is a derivative of the AND gate that represents a function where the output is off only if both inputs are present (Fig. 1E). An example of regulatory circuit endowed with an NAND behaviour consists of a strong promoter that is repressed by two weak TFs that act co-opera- tively to produce full repression [20]. A simpler way to produce a NAND-gate involves the action of a repressor as the first in- put and a small signal molecule (effector, co-repressor) as the second one. In the effector-free form, the repressor should have a weak inhibitory activity – or not repress at all. But then, in- ducer binding triggers a conformational change that increases the affinity of the repressor to the target sequence, turning the Fig. 2. Composite OR gates. The representation of operators and promoter activity off. Examples of this type include a large list promoter elements are the same of Fig. 1. (a) The NOR-gate is of regulators that bind DNA and inhibit transcription upon depicted as a promoter repressed by two TF. (b) One plausible ORN- binding an effector signal. The Fur (ferric uptake regulator) gate involves elements of the SOS system, where the target promoter is repressed by a TF (LexA) that is degraded by a free protein (the repressor is a good instance of this, as it strongly binds and protease RecA*, see text for explanation). (c) A XOR-gate can be blocks its target promoter sequences only when associated to assembled through the combination of two ANDN-gates with inverse divalent metal ions (Fe2+) [24]. A more intricate example of dependence of two separate TFs. (d) XNOR-gate is the result of NAND-gate is brought about by the dual XylR regulator of combining AND- and NOR-gates. the m-xylene degradation pathway from the TOL plasmid pWW0. In this example binding the aromatic inducer (m-xy- lene) makes the XylR protein a more efficient repressor of its where the gene expression takes place only when both input own Pr promoter [25,26]. signals are present (Fig. 1C). It is easy to visualize this gate Another logic operator derived from the AND gate is the in a cell either as a promoter that depends on two different so-called ANDN-gate (AND-NOT). Unlike the previous transcriptional activators acting co-operatively [20] or as a cases, the inputs are not equivalent and their order makes a transcriptional activator bound to an inducer molecule. This difference: the output is turned on only when the first input last setup is widespread among TFs that controls the expres- is present and the second is absent (Fig. 1F). In biological sion of microbial catabolic pathways for biodegradation of terms this gate can be materialized as a promoter that is acti- aromatic compounds [6]. vated by a TF (first input) and repressed by the other (second The OR-gate is the one where the output is on when one or input). In such a setup, if the repressor is bound to the pro- both inputs are high (Fig. 1D). This gate can be implemented moter, the activator is unable to act [2]. inter alia by a promoter that is activated alternatively by two Similarly to the derivatives of the AND gate just discussed, different TFs, as happens in many cases of cross-regulation there are four composite variants of the OR-gate (Fig. 2), [20,21]. The amplifier, NOT, AND and OR logic gates which impose a growing number of constraints on their poten- (Fig. 1A–D) are the most frequently accounted instances in tial biological counterparts of existing or artificial regulatory the biological literature, surely due to their simplicity and modules. The NOR-logical gate (NOT-OR) performs a func- widespread representation in many regulatory processes [2]. tion opposite to that of the OR-gate: the system is off when The AND- and OR-gates share two very important con- one or both inputs are present. A simple biological implemen- strains: (i) the need of a weak promoter to produce an off state tation of this gate could include a strong promoter that is in the absence of the inputs and (ii) the requirement of two regulated by two independent strong repressors (Fig. 2A). A activating inputs (two activators or one activator and one cog- second intriguing variant of the OR gate is the operator called nate inducer) to turn on promoter activity [20]. This makes ORN (OR-NOT, Fig. 2B), in which the output is off only when small changes in the binding affinities of the activators and the first input is high and the second is low, but remains on in the RNAP for the promoter region to convert one gate into any of the other 3 combinations. The added complexity of this another using the same regulatory modules [14]. For instance, gate relies in the requirement that the second input must shut- Mayo et al. [19] were able to create variants of the lac pro- down promoter activity in the absence of the first signal, but moter displaying AND and OR behaviours by introducing a the same second input should not inhibit the same promoter few point mutations in the CRP, LacI and RNAP binding sites when the first signal is present. Complex as it may look, the (six mutations were needed to produce the AND-gate and nine ORN-gate occurs naturally in promoters of the SOS response to generate an OR-gate [19]). This example (along with other to DNA damage mediated by RecA/LexA regulators. The 1240 R. Silva-Rocha, V. de Lorenzo / FEBS Letters 582 (2008) 1237–1244 salient feature of this system is that the proteolytic activity depends on the availability of robust regulatory modules that of RecA is activated by breaks in the DNA (caused by UV mimic Boolean gates. As discussed above, known natural pro- radiation, for example) that change the protein to its active karyotic transcriptional networks provide a wealth of such form (RecA*). This in turn causes the proteolytic cleavage modules that, either as they are or rationally reassembled, and degradation of the LexA repressor which otherwise inhi- are usable (but not optimal, see below) for such a circuit de- bits SOS promoters [27]. Promoters down-regulated by lexA sign. Yet, the number of actual regulatory setups available thus display a behaviour formally equivalent to an ORN-gate so far is limited [17,28]. In other cases, some of the most com- (Fig. 2B). plex logic gates have been simulated [2,20], but not yet found An additional modification of the OR-gate is the XOR oper- as naturally occurring regulatory schemes. This poses intrigu- ator (exclusively OR). In this gate, the system gives an on out- ing questions as to whether some of such regulatory arrange- put when one of the inputs is present, but not if both are either ments are possible but not biologically sustainable. Why? present or absent (Fig. 2C). The XOR-gate increases the com- Biological systems are permanently subject to Darwinian selec- plexity of its possible biological representations: the promoter tion and it may well happen that some of the synthetic regula- at stake is to be inactive in the absence of two signals, while it tory schemes do not nest well within the pre-existing must be switched on when one of them is present but off when regulatory network of the cell [29]. This is a general problem the two inputs coexist. Two possible biological constructs with of any implantation of heterologous genes or proteins in a cell XOR-gates have been proposed by Buchler et al. [20], one (see below). The intracellular molecular ecosystem displays a using a singular promoter and the other using two promoters. sort of resistance to colonization that is so typical, at a differ- Fig. 2C shows the two-promoter model of this XOR gate. This ent scale, of bacterial consortia [30]. One way of tackling this somewhat intricate arrangement involves the combination of important caveat is to survey the pool of existing, natural reg- two weak promoters with an ANDN-gate architecture but ulatory setups for desired input/output performances – rather with inverse dependences (i.e., one is A AND-NOT B and than forward-designing the same behaviours. One source of the other is B AND-NOT A). The rationale behind the design such assets is the whole of genetic circuits that control expres- is that the first weak promoter is activated by the first TF and sion of catabolic pathways for biodegradation of environmen- repressed by the second TF, while the second weak promoter is tal pollutants in soil bacteria [6]. These are often organized in repressed by the first TF and activated by the second TF. sub-networks full of regulators that respond to distinct chem- Under this arrangement, the presence of only one TF activates ical species, and cognate promoters endowed with the most di- one promoter and repress the other, thereby leading to the on verse connectivities. These systems are excellent starting state of one of the two, while the simultaneous presence or materials for mining and re-designing novel regulatory mod- absence of both TFs keeps both promoters silent (resulting ules. Furthermore, the growing ease of use of wet activity min- in an off state). A second proposal for a XOR gate involves ing methods for highly diverse environmental metagenomes one weak promoter controlled by two TFs which can either [31] allows the assembly of genetic traps in which a pool of gen- repress or activate depending on their binding to strategically omes is experimentally interrogated – and eventually the right positioned binding sites through the cognate DNA region [20]. clones selected – for promoters and regulators with given spec- Finally, Fig. 2D shows the XNOR gate (exclusively NOT- ificities and mode of functioning. One interesting approach to OR), also called EQU-logical operator. This gate is the oppo- this end was developed by WatanabeÕs group [32] for the site of the XOR-gate, as the output is on only if the input enrichment of metagenomic clones encoding promoters and signals are equal (low/low or high/high). Many different regulators responsive to aromatic hydrocarbons. This was schemes have been entertained for the implementation of a made by means of an operon-trap GFP-expression vector for biological XNOR-gate [2,20]. The scheme of Fig. 2D involves shotgun cloning that allowed for the selection of positive the use of two different promoters regulated by two dual (i.e. clones in liquid cultures by fluorescence-activated cell sorting. repressor–activator activity) TFs which interact cooperatively Other regulatory traps have been proposed as well for survey- in their activator facet – but not in their repressor function. ing metagenomes in vivo [33,34]. These can surely be further The first, weak promoter is cooperatively activated by the developed in the quest of more biologically significant equiva- two TFs (AND operator). The second promoter has a high lents to the Boolean gates analyzed above. activity but is strongly repressed separately by both TFs (NOR operator). Under this scheme, if only one input TF is present, both promoters are inactive (P needs both TFs and 3. De-compression and disambiguation of natural regulatory P0 is highly repressed by either TF). In the absence of any input modules TF, P is inactive and P0 is not repressed, thus providing an on output. But if both input TFs exist, P0 is repressed but P is As mentioned above, for the sake of circuit design promoter activated, resulting also in an on output. This combination performance can be abstracted to a limited number (ideally of promoters and TFs thus results in a factual XNOR-gate. one or two) of input functions and one output function. In There are others models for the construction of this same gate the real word, however, this behaviour is achieved only if the that imply the combination of looping-forming activators in rest of the many conditions that influence promoter function- complex promoter architectures with different binding sites ing in vivo (growth rate, temperature, physiological status, [20]. stress, etc.) are kept artificially constant. In other words, natu- rally occurring promoters, even the simplest, have always more 2.4. Mining building blocks for logic gates in the bacterial than two input functions. For artificial biological systems metagenome ambitioned to perform beyond the very limited conditions of Our ability to design biological circuits capable of imple- the Laboratory, this certainty cannot be ignored. Let us take, menting logic operations and even complex algorithms largely for instance, the Pu promoter encoded in the pWW0 TOL R. Silva-Rocha, V. de Lorenzo / FEBS Letters 582 (2008) 1237–1244 1241 plasmid for biodegradation of this aromatic hydrocarbon in Pseudomonas putida mt-2 ([35], Fig. 3). Pu belongs to the class of promoters that depend on the alternative sigma factor r54 and is activated at a distance by the m-xylene-responsive acti- vator (XylR). In principle, this is an ideal basis for developing a large number of AND and NAND gates, as XylR is also a m-xylene dependent repressor of its own Pr promoter [25,26]. Furthermore, XylR can be easily evolved to respond to non- natural aromatic effectors [36,37]. However, the action in vivo of XylR and m-xylene on Pu depends not only of these two inputs but also on a plethora of additional factors and sig- nals that tune promoter output to the general physiological and metabolic conditions of the cells [35]. Mechanistically, such signals are entered through the integration host factor (IHF), the IIANtr protein, through sigma factor competition, ppGpp levels, the TurA histone-like proteins and perhaps Fig. 3. Factors shown or suspect to influence the activity of the Pu other additional inputs as well (Fig. 3). The result of this is that promoter of plasmid pWW0. The r54 promoter Pu can be transcribed having the promoter turned on or off says little on the states of in vitro by just combining purified IHF, the sigma factor core RNAP the various inputs. In other words, the output is ambiguous and activated XylR. However, genetic analysis of mutants which affect and hardly usable for robust circuit design. To varying Pu activity in vivo, directed knockouts of genes known to affect other catabolic promoters and proteomic analysis of factors which bind the degrees, many other natural promoters are afflicted by the cognate DNA sequence have revealed a whole collection of additional same problem, as prokaryotic regulatory economy tends regulatory elements (proteins and small molecules) which influence to to compress control elements in growingly shorter DNA various degrees the outcome of Pu under diverse growth or stress sequences [38]. Fortunately, it is possible to solve such conditions. This regulatory compression (that is standard in many compressions and come up with promoters and genes with a prokaryotic promoters) makes difficult to use this promoter as an AND logic gate having solely XylR and m-xylene as inputs. Pu disambiguated performance. For instance, the sequence of mutants can however be produced that relieve the physiological the T7 phage genome has been altogether refactored for over- control and originate more orthogonal variants. coming regulatory compressions and produce biological parts amenable to robust engineering [38]. Similarly, Pu variants with stronger binding sequences for r54-containing RNAP not be examined in detail here. Yet, one emerging feature of have been built that overcome the physiological control of the intracellular organization of bacteria is worth considering transcription [39] and thus make the regulatory module at this point. Namely, the association of many, perhaps most XylR/m-xylene/Pu a robust AND gate. The issue at stake, polypeptides into multi-protein structures [40,41] and the likely however, is whether such improved regulatory and genetic role many orphan genes products to scaffold enzymatic com- blocks will be stable enough on the long run or they will re- plexes [42,43]. In particular, it seems that most proteins are lapse into compression and ambiguity as soon as they are ex- not singular objects let loose in the cytoplasm, but pieces of posed to Darwinian selection. multi-part, dynamic structures that assemble through a cons- tellation of specific interactions [40]. Furthermore, there are indications that functionally related gene clusters or genomic islands are located in distinct places of the chromosome that 4. Orthogonalization or nesting? Dealing with system constraints target their transcription to given spots of the cytoplasm and boundaries [44]. This means that each protein needs to be expressed and located in an intracellular physical niche for optimally per- Since bacteria lack intracellular compartments comparable forming its function. Polypeptides unable to fit within such to their eukaryotic counterparts it is tempting to make the assemblies might be rendered not functional and eventually re- abstraction of them as bags of enzymes. In such a scenario, jected through a simple Darwinian mechanism. While inserting the various macromolecules, proteins and metabolites could extra DNA in a cell (artificially or naturally through horizon- move freely through the whole available space. It is also stan- tal gene transfer) may per se be limited only by the restriction dard to represent the DNA as a line (a circle for whole gen- systems of the recipient, eventual implantation of the encoded omes) in which genes are duly assembled in one dimension proteins might be severely counterselected. This notion is sug- and read once at a time by the expression machinery, following gested not only by the long period of time that horizontally the instructions encoded by the same sequence. Useful as this transferred genes take to develop regulatory interactions [29] picture may have been in the early times of Molecular Genet- but also by the resistance to transfer of genes whose products ics, it is altogether insufficient to understand how whole cells belong to multiprotein complexes [45]. The practical downside and whole genomes work. The advent of genomic and proteo- of these biological phenomena is the difficulty to program bac- mic approaches to study whole-cell prokaryotic biology is teria with genetic circuits or otherwise implemented through revealing a large number of constraints that limit the perfor- heterologous expression of regulatory modules. mance of any molecular machinery – let alone one implanted Is there a way to escape this apparently unsurmountable artificially, within the overcrowded volume of a bacterium. constraint? The question is still open at the time of writing this The different types of constraints and their consequences for article. Every synthetic circuit engineered so far in bacteria to designing artificial biological systems – including artificial behave in a particular way seems to decay rapidly after a cells – have been brilliantly reviewed by Danchin [3] and will relatively short period of performing time [18,46]. The one 1242 R. Silva-Rocha, V. de Lorenzo / FEBS Letters 582 (2008) 1237–1244 solution in sight may come from the way natural mobile regu- latory circuits (like those present in phages, transposons and broad host range plasmids [47] succeed in functioning in their new hosts). Fig. 4 sketches what appear to be the two basic evolutionary strategies to this end. In one case, the mobile DNA is associated to error-prone DNA polymerases [48] which may help to generate enough diversity through the transferred protein sequences as to facilitate their nesting in the recipient protein network [35]. This evolutionary device could be recreated in the laboratory by endowing biological parts with a superior evolvability (see for instance [49] as a pos- sible technique to this end). In the second case, the proteins and functions encoded by the transferred DNA appear to be evolutionarily selected for lacking any dependence on the bio- Fig. 4. Evolutionary strategies of implantation of new proteins within existing functional complexes. Cellular proteins are often placed in logical context of the recipient. The quality of behaving in a large multicomponent complexes with defined – although dynamic – 1 context-free fashion is designed as orthogonality, echoing architectures brought about by surface interactions between the equivalent properties in computing science. Examples of natu- constituents. The entry of a new protein lacking adequate contact ral orthogonal parts include the T7 phage polymerase (able to surfaces in such a setting might result in the loss of function in vivo. One stratagem to overcome this setback is to increase the mutation transcribe genes under the T7 promoter sequence in basically rate of the DNA sequence involved and produce enough diversity and any biological host) and the broad host range promoters of selection of variants for reconstruction of the required interactions. A bacterial integrons [50] that promote recruitment of antibiotic second possibility for the new protein is to evolve as an orthogonal resistance genes to promiscuous plasmids. There is a good deal component, the activity of which is altogether context-independent. of information and actual parts to be mined from the wealth of See text for discussion. data on promiscuous bacterial functions (broad host range promoters, antibiotic resistance genes, integrons and the like). In the meantime, artificial orthogonal biological systems have been already developed. One impressive achievement in this re- But, despite the various control modules available for spect is the generation of multiple orthogonal ribosome/ constructing biological networks, the regulatory landscape of mRNA pairs that can process information in parallel with, the prokaryotic metagenome remains to be explored for new but independent of, their wild-type counterparts [51]. In these gates and components. Much of the current challenge for pairs, the orthogonal ribosome exclusively translates the robust design deals with the upgrade of naturally existing orthogonal mRNA, which is not a substrate for the other promoter/regulator setups to fulfill the requirement of con- housekeeping ribosomes. Both mining of naturally occurring text-independence that is necessary for vigorous engineering orthogonal systems and design of artificial context-indepen- of biological systems. However, context-dependency, regula- dent biological functions will surely improve the robustness tory compression and Darwinian evolvability seem to be of artificial genetic circuits to a point much closer to the per- intrinsic to any component of existing live cells. It is thus likely formance of equivalent electronic setups. that the straight adoption of the jargon and conceptual frames of electric and mechanical does not suffice to move the field satisfactorily forward. The research agenda of Synthetic Biol- 5. Outlook ogy for the next few years may thus include not only a focus to identify the functional objects and modules (regulatory or Logic gates based on prokaryotic promoters provide an otherwise) that result from de-constructing live systems (such attractive frame for designing artificial biological circuits. As as prokaryotic cells), but also the mapping and understanding shown above, a virtually unlimited diversity of schemes can the relationships between such modules [3]. Moreover, if the be produced by just combining a limited number of modules: ambition is to produce engineered biological systems to per- weak or strong promoters, activators and repressors, and dual form over time, one must consider mutation and evolutionary TF with or without cooperative activity. Owing to their selection as a perpetual working condition. This poses a num- response to a large variety of chemical inducers, regulatory ber of fundamental questions on whether we can program elements of catabolic operons for xenobiotic molecules found genes to be more or less evolvable, and their functions more in soil bacteria become attractive sources for this type of circuit or less orthogonal. Eventually, the question becomes whether building blocks [6,52]. TF of this sort include members of the an alternative coding molecule (and the corresponding expres- LysR, IclR, AraC/XylS, XylR/NtrC, TetR, GntR, MarR sion machinery) can be produced that is not as amenable to families [52], which display a large repertory of molecular mutation as DNA. While this is not yet in sight [54], we can mechanisms (repression, activation, loop-formation, dual still attempt the development of partially orthogonal systems regulation, cross-regulation) to execute their regulatory func- or, alternatively, benefit from mutation and evolvability along tion. Many of these TFs and promoters can further be evolved the lines discussed above. in their effector specificities and DNA binding sites [53] thereby expanding the repertoire of assets for circuit design. Acknowledgements: Authors are indebted to A. Danchin, S. Panke, V. dos Santos, J. Poyatos and J.L. Martı´nez for inspiring discussions. The work in AuthorsÕ Laboratory is supported by contracts of the Frame- 1A is orthogonal to B if A does not influence B. Orthogonality work Programmes of the EU and by grants of Spanish Ministry of guarantees that changes made in a component of a system neither Education and Science. This review was made under the auspices of creates nor propagates side effects to other components of the system. the european networks EMERGENCE and TARPOL. R. Silva-Rocha, V. de Lorenzo / FEBS Letters 582 (2008) 1237–1244 1243

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