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Expanding the synthetic biology toolbox: engineering orthogonal regulators of gene expression
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Expanding the synthetic biology toolbox: engineering orthogonal regulators of gene expression Christopher V Rao Despite substantial progress in synthetic biology, we still lack the ability to engineer anything as complex as Nature has. One of the many reasons is that we lack the requisite tools for independently controlling the expression of multiple genes in parallel. While our toolbox is still spare, the situation is rapidly changing. This opinion discusses some recent approaches and open challenges in designing orthogonal regulators of gene expression in bacteria. Address Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States Corresponding author: Rao, Christopher V ([email protected])
Current Opinion in Biotechnology 2012, 23:689–694 This review comes from a themed issue on Tissue, cell and pathway engineering Edited by Hal Alper and Wilfried Weber For a complete overview see the Issue and the Editorial Available online 9th January 2012 0958-1669/$ – see front matter, Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.copbio.2011.12.015
Introduction Life is complex. Even the simplest living organisms are comprised of hundreds, if not many thousands of interacting proteins, nucleic acids, lipids, and metabolites. Yet when one considers most examples in synthetic biology, simplicity becomes the operative word – most designs involve no more than a handful of interacting components. Clearly, substantial progress has been made using relatively simple designs. However, we are still far away from engineering anything as complex as to what is observed in nature. Quite simply, our toolbox is still too primitive. We lack, in particular, the ability to independently control multiple cellular processes in parallel. As an example, consider the tools available for independently and reliably modulating the transcription of multiple genes in the model bacterium Escherichia coli. If one enumerates them, then the list numbers roughly half a dozen (a number nonetheless far greater than in many other organisms) [1]. Contrast this number with the native regulatory machinery of E. coli, where the number of transcription factors alone exceeds 300 [2]. www.sciencedirect.com
A further limitation is that many of these tools coop the native regulatory machinery of the cell rather than function independently of it. Many popular expression systems, such as the ones based on LacI or AraC for example, duplicate existing regulatory mechanisms within the cell rather than function independently of them. While in some cases, this regulatory crosstalk may be insignificant or easily rectified [3,4]. In other cases, it may be more problematic particularly with more complex and expansive designs, where the temptation will be to coop additional, native regulators. Ideally, the synthetic biologist would possess hundreds of regulators for independently modulating not only the transcription and translation of a given gene but also the activity of the associated gene product [1,5]. She should also be able to couple these regulators to different intercellular and intracellular signals, much like what occurs in cells. In addition, she would not be restricted to a particular mechanism such as transcriptional repression but instead have a full menu of options at her disposal. Lastly, she would possess tools that are orthogonal not only to existing cellular processes but also one another. This opinion piece discusses some recent advances in expanding the synthetic biology toolbox for prokaryotes, E. coli in particular. As a number of excellent and more expansive reviews have already been recently written on the topic (e.g. [6,7]), the focus here is on the issue of orthogonally (Figure 1). In the remainder, we discuss some recent approaches and future challenges towards engineering orthogonal regulators of gene expression.
Engineering orthogonal regulators of transcription Most examples in synthetic biology involve a gene regulatory circuit where the primary logical functions occur at the level of transcription. As we have already mentioned, the transcriptional toolbox is surprising limited in bacteria given that transcription is the best-understood regulatory mechanism in these cells. Expanding it will be a critical step in designing more complicated gene circuits. The most successful approach for expanding the transcriptional toolbox has been to introduce transcription factors from different species, phage, and transposable DNA elements. Notable examples include TetR from transposon Tn10, cI from bacteriophage Lambda, and LuxR from Vibrio fischeri. When these transcription factors Current Opinion in Biotechnology 2012, 23:689–694
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Figure 1
Figure 2
Signal A
Gene A
Signal B
Gene B Current Opinion in Biotechnology
Two regulators are orthogonal if their signals and targets are distinct. When crosstalk occurs between two regulators, denoted by the gray arrows, one can no longer independently control the expression of the two genes. Expanding the number of orthogonal regulators will enable to design of more complex networks. Different approaches for engineering orthogonal regulation in bacteria are discussed in this opinion piece.
are taken from different organisms such as in the case of LuxR, the potential for crosstalk is minimal as the donor organism evolved independently of the target one. The situation is less clear with phage and other transmissible DNA elements that co-evolve with their hosts. Lambda cI, for example, is known to directly repress the transcription of the pckA gene in E. coli [8], an interaction difficult to predict ahead of time. Despite the potential for crosstalk with host genes, this approach is still the most promising, as the pool of candidate transcription factors is nearly limitless. With rapid advances in DNA sequencing and synthesis, one can easily envision assembling vast libraries of orthogonal transcription factor by direct synthesis. The main challenge will be to characterize them all. In many cases, these transcription factors will be orphans – not only will their target binding sequence not be known but also their activating signals (most bacterial transcription factors form one-component signal transduction systems [9]). Determining both will require laborious screens. The main reason why foreign transcription factors such as TetR and LuxR are so popular is that they have previously been characterized. An alternate approach is to mimic the evolutionary process itself. Complex gene networks are thought to have evolved primarily by gene duplication [10]. The same process can also potentially be employed in synthetic biology (Figure 2). Hillen and colleagues, for example, employed such an approach to engineer a system for the orthogonal control of gene expression using two TetR variants: one that selectivity bound a new DNA sequence Current Opinion in Biotechnology 2012, 23:689–694
Current Opinion in Biotechnology
Gene networks are thought to evolve primarily through gene duplication events [10]. In the simple example depicted in this cartoon, a single transcription factors, depicted by a red, regulates the expression of a single gene, also given in red. Following a duplication event, two transcription factors, depicted by the red and purple circles, now regulate the expression of the red gene. During subsequent divergence, one of the transcription factors, denoted by the red circle, can regulate a new gene, given in blue. Such a process is thought to provide one mechanism for the evolution of gene networks. A similar process can be employed when engineering synthetic networks.
and another that was activated by a new chemical [11]. Using these two variants, they were able to independently control the expression of two genes. The advantage of this approach is that it starts with a wellcharacterized transcription factor and then uses it as a scaffold to engineer new ones. The same approach could be applied to other transcription factors. The main challenge is to identify these specificity-altering mutations [12]. In principle, one could exploit the modularity of bacterial transcription factors by swapping different subdomains between them [13,14]. In practice, however, this strategy does not appear to work very well in general and is limited to closely related transcription factors. A more promising approach is to use directed evolution [12,15]. In the case of ligand specificity, a number of researchers have been able to engineer transcription factors that no longer respond to their native signal but instead to entirely new and distinct ones [16–20]. However, the extent to which the specificity can be changes appears to be limited to chemically similar compounds [12]. Similar approaches can also be applied to alter the DNAbinding specificity of a transcription factor, though many www.sciencedirect.com
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of the same problems remain. To bypass some of difficulties associated with identifying these mutations, we recently employed comparative genomics to explore how the CRP/FNR family of transcription factors co-evolved with their target DNA-binding sequence [21]. From this analysis, we were able to engineer a number of CRP variants that bind a new target sequence and not their native one. While these variants were generally orthogonal to one another, a number of unanticipated interactions were nonetheless observed. Similar analysis has been performed for the LacI family of transcription factors though the commensurate experiments have yet to be performed [22]. While this approach is promising, it still provides an incomplete description of the specificitydetermining residues within these proteins. One additional challenge meriting discussion is that many bacterial transcription factors are homodimers, and different variants may form heterodimers with one another [23]. In the TetR example discussed above, Hillen and coworkers were able to engineer these proteins so that they form homodimers. Alternate solutions are to engineer single-chain transcription factors [24] or work with monomeric ones such as MarA [25]. Suffice to say, engineering bacterial transcription factors is still a challenging problem. This situation is not so bleak in eukaryotes. Artificial transcription factors based on polydactyl zinc fingers and TAL effectors offer powerful approaches for expanding the synthetic biology toolbox [26,27]. The modularity of these DNA-binding elements enables one to engineer proteins that target specific DNA sequences. In addition, these engineered DNA-binding proteins can be fused to different effector domains, enabling one to regulate the transcription of potentially any genes. Whether the same approaches can be employed in bacteria is unknown. While there have been reports of using polydactyl zinc fingers to regulate gene expression in E. coli [28], their utility appears somewhat limited. Nonetheless, this is an avenue meriting further investigation.
Orthogonal regulation of translation using orthogonal ribosomes The greatest advances to date have been made with RNAbased regulation. We comment only briefly on these tools as they recently have been the subject of a number of comprehensive reviews [29–31]. Briefly, these engineered regulators target translation through the use of cis and transacting RNA modules [32–35]. The strength of these RNAbased designs is that they are easily scalable in an orthogonal manner and that the design rules are somewhat transparent, unlike the case with proteins. In addition, these regulators can be allosterically regulated with small molecules by coupling them to ribozyme switches. RNA clearly provides a more facile route for engineering orthogonal regulators of gene expression then proteins. One www.sciencedirect.com
immediate question then is whether this is better. The author is not in a position to answer this question. That said, the two are not distinct. For one, RNA-based regulators still function downstream of transcription and thus are dependent on our ability to control their expression. In addition, a number of theoretical studies suggest that proteins and RNAs are suited for different regulatory tasks [36,37]. This would suggest that the two are best used as complementary tools rather than as substitutes for one another.
Engineering orthogonal regulation of translation using orthogonal ribosomes Regulators based on sRNA’s are not the only tools available for regulating translating – orthogonal ribosomes (oribosomes) are another. Briefly, o-ribosomes translate genes with altered Shine-Dalgarno sequences not recognized by host ribosomes. The advantage of using o-ribosomes is that they partially decouple the translation of a target gene from the native translational machinery in the cell. While orthogonal ribosomes are not new [38], previous designs were inefficient and often toxic to the cell [39,40]. More recently, Rackham and Chin developed a powerful dual-selection procedure for isolating o-ribosomes that bypass many of these previous limitations [41]. In particular, their designs were able to efficiently translate non-cognate mRNA without any toxic affects. Moreover, they were able to isolate multiple o-ribosomes/ mRNA pairs that are not only orthogonal to host ones but also other o-ribosome/mRNA pairs. An and Chin extended this concept to synthetize networks where both transcription and translation were decoupled from the host systems using a design by coupling their o-ribosomes to a T7 expression system [42]. Motivated by the results of Rackham and Chin, we recently developed a computational procedure for engineering oribosomes working under the assumption that base-pair interactions between the 16S rRNA and the 50 untranslated region of mRNA serve as the primary mode for ribosome binding and translational initiation [43]. Using this algorithm, we were able to similarly engineer a number of oribosomes in E. coli, thereby bypassing a tedious selection procedure. This work further illustrates the power of RNAbased regulators where design rules based on complementary base-pair interactions are easily distilled. Similar strategies have also been applied transcription. Here, researchers have designed circuits based on T3 and T7 expression systems [44,45]. Interestingly, researchers have been able to alter the promoter specificity for these phage polymerases [46], suggesting that orthogonal polymerases can also be engineered.
Engineering orthogonal regulation using twocomponent signal transduction system Two-component systems are signal transduction modules commonly employed in bacteria [47] (Figure 3). The Current Opinion in Biotechnology 2012, 23:689–694
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basic structure consists of a transmembrane sensor kinase that regulates phosphorylation state of a soluble response regulator, which is commonly a transcription factor. A number of designs in synthetic biology have employed two-component systems [48,49,50]. Many involve chimeras based on the EnvZ sensor kinase: by fusing different signaling domains to the kinase domain of EnvZ, researcher have been able to construct sensors responsive to different amino acids [51], sugars [52,53], and most impressively light [54]. Two-components systems are highly specific with minimal crosstalk between different pairs of cognate sensor kinases and response regulators [55]. Because of their native specificity, two-component systems are ideal tools for orthogonal gene expression. As an example, Voigt and co-workers engineered a system for differentially controlling gene expression using two different wavelengths of light [50]. Ideally, one could alter the specificity of these
sensor kinases so that different stimuli could be used to regulate alternate response regulators [56]. Towards this goal, a number of researchers have recently employed comparative genomics to identify the specificity determining residues between different cognate sensor kinase/ response regulator pairs [57–58,59]. Laub and co-workers applied this analysis to identify mutations that alter the specificity of the sensor kinases for their target response regulators [59]. Armitage and co-workers, on the contrary, were able to change the specificity of sensor kinase using structural analysis [60]. More recently, Goulian and coworkers employed directed evolution to make these engineered bifunctional sensor kinase/response regulator pairs more responsive to their input signals [61]. These results are all very promising as they offer an entirely new route for orthogonal gene regulation, namely by changing how these proteins interact with one another.
Conclusions While the synthetic biology toolbox is still quite limited, substantial progress towards expanding it has been made during the past few years. What is remarkable about these advances is their breadth. While the transcriptional toolbox is still sparse, this is not the case when one considers post-transcriptional regulation, particularly RNA-based designs targeting translation. This indicates a new paradigm is emerging in synthetic biology, one away from designs based solely on transcriptional regulation and towards one embracing the full complexity of the cell.
Figure 3
Signal A
Signal B
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Gene A
P
Gene B Current Opinion in Biotechnology
The canonical structure of a two-component system is a transmembrane sensor kinase linked to a soluble response regulator, respectively depicted as an oval and a circle. The sensor kinase regulates the phosphorylation state of the response regulator. Typically, the response regulator is active only when phosphorylated. Aside from regulating transcription, response regulators can also control other processes, motility being the prime example. One advantage of using twocomponent systems is that the interaction between sensor kinase and response regulator is exquisitely specific. Current Opinion in Biotechnology 2012, 23:689–694
Interestingly, an alternate paradigm has recently emerged that may bypass some limitations associated with a limited toolbox. In particular, a number of analogies have been made between synthetic biology and electrical circuit design. While this analogy may be apt in some regards, it is clearly lacking in others. Biological circuits involve thousands of distinct components whereas in electrical circuits involve just a few. The reason, of course, is that the components in electrical circuits are physically separated from one another, enabling them to be reused without the potential for unwarranted crosstalk. This same strategy has recently been applied in synthetic biology [62,63]. Rather than engineer networks within cells, these designs form them between cells. How scalable this approach is remains unknown. Nonetheless, it provides an intriguing route for engineering complex synthetic networks. We conclude by noting that orthogonal regulation is not solely a synthetic biology cause but one that extends to biology in general. The ability to independently control the expression and activity of multiple genes in parallel will greatly enable not only the engineering of synthetic networks but also the reverse engineering of native ones. In these regards, the aims of synthetic biology are not orthogonal to those in traditional biology. www.sciencedirect.com
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Acknowledgements CVR acknowledges support from the National Science Foundation and is affiliated with the Energy Biosciences Institute.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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Expanding the synthetic biology toolbox: engineering orthogonal regulators of gene expression Christopher V Rao Despite substantial progress in synthetic biology, we still lack the ability to engineer anything as complex as Nature has. One of the many reasons is that we lack the requisite tools for independently controlling the expression of multiple genes in parallel. While our toolbox is still spare, the situation is rapidly changing. This opinion discusses some recent approaches and open challenges in designing orthogonal regulators of gene expression in bacteria. Address Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States Corresponding author: Rao, Christopher V ([email protected])
Current Opinion in Biotechnology 2012, 23:689–694 This review comes from a themed issue on Tissue, cell and pathway engineering Edited by Hal Alper and Wilfried Weber For a complete overview see the Issue and the Editorial Available online 9th January 2012 0958-1669/$ – see front matter, Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.copbio.2011.12.015
Introduction Life is complex. Even the simplest living organisms are comprised of hundreds, if not many thousands of interacting proteins, nucleic acids, lipids, and metabolites. Yet when one considers most examples in synthetic biology, simplicity becomes the operative word – most designs involve no more than a handful of interacting components. Clearly, substantial progress has been made using relatively simple designs. However, we are still far away from engineering anything as complex as to what is observed in nature. Quite simply, our toolbox is still too primitive. We lack, in particular, the ability to independently control multiple cellular processes in parallel. As an example, consider the tools available for independently and reliably modulating the transcription of multiple genes in the model bacterium Escherichia coli. If one enumerates them, then the list numbers roughly half a dozen (a number nonetheless far greater than in many other organisms) [1]. Contrast this number with the native regulatory machinery of E. coli, where the number of transcription factors alone exceeds 300 [2]. www.sciencedirect.com
A further limitation is that many of these tools coop the native regulatory machinery of the cell rather than function independently of it. Many popular expression systems, such as the ones based on LacI or AraC for example, duplicate existing regulatory mechanisms within the cell rather than function independently of them. While in some cases, this regulatory crosstalk may be insignificant or easily rectified [3,4]. In other cases, it may be more problematic particularly with more complex and expansive designs, where the temptation will be to coop additional, native regulators. Ideally, the synthetic biologist would possess hundreds of regulators for independently modulating not only the transcription and translation of a given gene but also the activity of the associated gene product [1,5]. She should also be able to couple these regulators to different intercellular and intracellular signals, much like what occurs in cells. In addition, she would not be restricted to a particular mechanism such as transcriptional repression but instead have a full menu of options at her disposal. Lastly, she would possess tools that are orthogonal not only to existing cellular processes but also one another. This opinion piece discusses some recent advances in expanding the synthetic biology toolbox for prokaryotes, E. coli in particular. As a number of excellent and more expansive reviews have already been recently written on the topic (e.g. [6,7]), the focus here is on the issue of orthogonally (Figure 1). In the remainder, we discuss some recent approaches and future challenges towards engineering orthogonal regulators of gene expression.
Engineering orthogonal regulators of transcription Most examples in synthetic biology involve a gene regulatory circuit where the primary logical functions occur at the level of transcription. As we have already mentioned, the transcriptional toolbox is surprising limited in bacteria given that transcription is the best-understood regulatory mechanism in these cells. Expanding it will be a critical step in designing more complicated gene circuits. The most successful approach for expanding the transcriptional toolbox has been to introduce transcription factors from different species, phage, and transposable DNA elements. Notable examples include TetR from transposon Tn10, cI from bacteriophage Lambda, and LuxR from Vibrio fischeri. When these transcription factors Current Opinion in Biotechnology 2012, 23:689–694
690 Tissue, cell and pathway engineering
Figure 1
Figure 2
Signal A
Gene A
Signal B
Gene B Current Opinion in Biotechnology
Two regulators are orthogonal if their signals and targets are distinct. When crosstalk occurs between two regulators, denoted by the gray arrows, one can no longer independently control the expression of the two genes. Expanding the number of orthogonal regulators will enable to design of more complex networks. Different approaches for engineering orthogonal regulation in bacteria are discussed in this opinion piece.
are taken from different organisms such as in the case of LuxR, the potential for crosstalk is minimal as the donor organism evolved independently of the target one. The situation is less clear with phage and other transmissible DNA elements that co-evolve with their hosts. Lambda cI, for example, is known to directly repress the transcription of the pckA gene in E. coli [8], an interaction difficult to predict ahead of time. Despite the potential for crosstalk with host genes, this approach is still the most promising, as the pool of candidate transcription factors is nearly limitless. With rapid advances in DNA sequencing and synthesis, one can easily envision assembling vast libraries of orthogonal transcription factor by direct synthesis. The main challenge will be to characterize them all. In many cases, these transcription factors will be orphans – not only will their target binding sequence not be known but also their activating signals (most bacterial transcription factors form one-component signal transduction systems [9]). Determining both will require laborious screens. The main reason why foreign transcription factors such as TetR and LuxR are so popular is that they have previously been characterized. An alternate approach is to mimic the evolutionary process itself. Complex gene networks are thought to have evolved primarily by gene duplication [10]. The same process can also potentially be employed in synthetic biology (Figure 2). Hillen and colleagues, for example, employed such an approach to engineer a system for the orthogonal control of gene expression using two TetR variants: one that selectivity bound a new DNA sequence Current Opinion in Biotechnology 2012, 23:689–694
Current Opinion in Biotechnology
Gene networks are thought to evolve primarily through gene duplication events [10]. In the simple example depicted in this cartoon, a single transcription factors, depicted by a red, regulates the expression of a single gene, also given in red. Following a duplication event, two transcription factors, depicted by the red and purple circles, now regulate the expression of the red gene. During subsequent divergence, one of the transcription factors, denoted by the red circle, can regulate a new gene, given in blue. Such a process is thought to provide one mechanism for the evolution of gene networks. A similar process can be employed when engineering synthetic networks.
and another that was activated by a new chemical [11]. Using these two variants, they were able to independently control the expression of two genes. The advantage of this approach is that it starts with a wellcharacterized transcription factor and then uses it as a scaffold to engineer new ones. The same approach could be applied to other transcription factors. The main challenge is to identify these specificity-altering mutations [12]. In principle, one could exploit the modularity of bacterial transcription factors by swapping different subdomains between them [13,14]. In practice, however, this strategy does not appear to work very well in general and is limited to closely related transcription factors. A more promising approach is to use directed evolution [12,15]. In the case of ligand specificity, a number of researchers have been able to engineer transcription factors that no longer respond to their native signal but instead to entirely new and distinct ones [16–20]. However, the extent to which the specificity can be changes appears to be limited to chemically similar compounds [12]. Similar approaches can also be applied to alter the DNAbinding specificity of a transcription factor, though many www.sciencedirect.com
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of the same problems remain. To bypass some of difficulties associated with identifying these mutations, we recently employed comparative genomics to explore how the CRP/FNR family of transcription factors co-evolved with their target DNA-binding sequence [21]. From this analysis, we were able to engineer a number of CRP variants that bind a new target sequence and not their native one. While these variants were generally orthogonal to one another, a number of unanticipated interactions were nonetheless observed. Similar analysis has been performed for the LacI family of transcription factors though the commensurate experiments have yet to be performed [22]. While this approach is promising, it still provides an incomplete description of the specificitydetermining residues within these proteins. One additional challenge meriting discussion is that many bacterial transcription factors are homodimers, and different variants may form heterodimers with one another [23]. In the TetR example discussed above, Hillen and coworkers were able to engineer these proteins so that they form homodimers. Alternate solutions are to engineer single-chain transcription factors [24] or work with monomeric ones such as MarA [25]. Suffice to say, engineering bacterial transcription factors is still a challenging problem. This situation is not so bleak in eukaryotes. Artificial transcription factors based on polydactyl zinc fingers and TAL effectors offer powerful approaches for expanding the synthetic biology toolbox [26,27]. The modularity of these DNA-binding elements enables one to engineer proteins that target specific DNA sequences. In addition, these engineered DNA-binding proteins can be fused to different effector domains, enabling one to regulate the transcription of potentially any genes. Whether the same approaches can be employed in bacteria is unknown. While there have been reports of using polydactyl zinc fingers to regulate gene expression in E. coli [28], their utility appears somewhat limited. Nonetheless, this is an avenue meriting further investigation.
Orthogonal regulation of translation using orthogonal ribosomes The greatest advances to date have been made with RNAbased regulation. We comment only briefly on these tools as they recently have been the subject of a number of comprehensive reviews [29–31]. Briefly, these engineered regulators target translation through the use of cis and transacting RNA modules [32–35]. The strength of these RNAbased designs is that they are easily scalable in an orthogonal manner and that the design rules are somewhat transparent, unlike the case with proteins. In addition, these regulators can be allosterically regulated with small molecules by coupling them to ribozyme switches. RNA clearly provides a more facile route for engineering orthogonal regulators of gene expression then proteins. One www.sciencedirect.com
immediate question then is whether this is better. The author is not in a position to answer this question. That said, the two are not distinct. For one, RNA-based regulators still function downstream of transcription and thus are dependent on our ability to control their expression. In addition, a number of theoretical studies suggest that proteins and RNAs are suited for different regulatory tasks [36,37]. This would suggest that the two are best used as complementary tools rather than as substitutes for one another.
Engineering orthogonal regulation of translation using orthogonal ribosomes Regulators based on sRNA’s are not the only tools available for regulating translating – orthogonal ribosomes (oribosomes) are another. Briefly, o-ribosomes translate genes with altered Shine-Dalgarno sequences not recognized by host ribosomes. The advantage of using o-ribosomes is that they partially decouple the translation of a target gene from the native translational machinery in the cell. While orthogonal ribosomes are not new [38], previous designs were inefficient and often toxic to the cell [39,40]. More recently, Rackham and Chin developed a powerful dual-selection procedure for isolating o-ribosomes that bypass many of these previous limitations [41]. In particular, their designs were able to efficiently translate non-cognate mRNA without any toxic affects. Moreover, they were able to isolate multiple o-ribosomes/ mRNA pairs that are not only orthogonal to host ones but also other o-ribosome/mRNA pairs. An and Chin extended this concept to synthetize networks where both transcription and translation were decoupled from the host systems using a design by coupling their o-ribosomes to a T7 expression system [42]. Motivated by the results of Rackham and Chin, we recently developed a computational procedure for engineering oribosomes working under the assumption that base-pair interactions between the 16S rRNA and the 50 untranslated region of mRNA serve as the primary mode for ribosome binding and translational initiation [43]. Using this algorithm, we were able to similarly engineer a number of oribosomes in E. coli, thereby bypassing a tedious selection procedure. This work further illustrates the power of RNAbased regulators where design rules based on complementary base-pair interactions are easily distilled. Similar strategies have also been applied transcription. Here, researchers have designed circuits based on T3 and T7 expression systems [44,45]. Interestingly, researchers have been able to alter the promoter specificity for these phage polymerases [46], suggesting that orthogonal polymerases can also be engineered.
Engineering orthogonal regulation using twocomponent signal transduction system Two-component systems are signal transduction modules commonly employed in bacteria [47] (Figure 3). The Current Opinion in Biotechnology 2012, 23:689–694
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basic structure consists of a transmembrane sensor kinase that regulates phosphorylation state of a soluble response regulator, which is commonly a transcription factor. A number of designs in synthetic biology have employed two-component systems [48,49,50]. Many involve chimeras based on the EnvZ sensor kinase: by fusing different signaling domains to the kinase domain of EnvZ, researcher have been able to construct sensors responsive to different amino acids [51], sugars [52,53], and most impressively light [54]. Two-components systems are highly specific with minimal crosstalk between different pairs of cognate sensor kinases and response regulators [55]. Because of their native specificity, two-component systems are ideal tools for orthogonal gene expression. As an example, Voigt and co-workers engineered a system for differentially controlling gene expression using two different wavelengths of light [50]. Ideally, one could alter the specificity of these
sensor kinases so that different stimuli could be used to regulate alternate response regulators [56]. Towards this goal, a number of researchers have recently employed comparative genomics to identify the specificity determining residues between different cognate sensor kinase/ response regulator pairs [57–58,59]. Laub and co-workers applied this analysis to identify mutations that alter the specificity of the sensor kinases for their target response regulators [59]. Armitage and co-workers, on the contrary, were able to change the specificity of sensor kinase using structural analysis [60]. More recently, Goulian and coworkers employed directed evolution to make these engineered bifunctional sensor kinase/response regulator pairs more responsive to their input signals [61]. These results are all very promising as they offer an entirely new route for orthogonal gene regulation, namely by changing how these proteins interact with one another.
Conclusions While the synthetic biology toolbox is still quite limited, substantial progress towards expanding it has been made during the past few years. What is remarkable about these advances is their breadth. While the transcriptional toolbox is still sparse, this is not the case when one considers post-transcriptional regulation, particularly RNA-based designs targeting translation. This indicates a new paradigm is emerging in synthetic biology, one away from designs based solely on transcriptional regulation and towards one embracing the full complexity of the cell.
Figure 3
Signal A
Signal B
P
Gene A
P
Gene B Current Opinion in Biotechnology
The canonical structure of a two-component system is a transmembrane sensor kinase linked to a soluble response regulator, respectively depicted as an oval and a circle. The sensor kinase regulates the phosphorylation state of the response regulator. Typically, the response regulator is active only when phosphorylated. Aside from regulating transcription, response regulators can also control other processes, motility being the prime example. One advantage of using twocomponent systems is that the interaction between sensor kinase and response regulator is exquisitely specific. Current Opinion in Biotechnology 2012, 23:689–694
Interestingly, an alternate paradigm has recently emerged that may bypass some limitations associated with a limited toolbox. In particular, a number of analogies have been made between synthetic biology and electrical circuit design. While this analogy may be apt in some regards, it is clearly lacking in others. Biological circuits involve thousands of distinct components whereas in electrical circuits involve just a few. The reason, of course, is that the components in electrical circuits are physically separated from one another, enabling them to be reused without the potential for unwarranted crosstalk. This same strategy has recently been applied in synthetic biology [62,63]. Rather than engineer networks within cells, these designs form them between cells. How scalable this approach is remains unknown. Nonetheless, it provides an intriguing route for engineering complex synthetic networks. We conclude by noting that orthogonal regulation is not solely a synthetic biology cause but one that extends to biology in general. The ability to independently control the expression and activity of multiple genes in parallel will greatly enable not only the engineering of synthetic networks but also the reverse engineering of native ones. In these regards, the aims of synthetic biology are not orthogonal to those in traditional biology. www.sciencedirect.com
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Acknowledgements CVR acknowledges support from the National Science Foundation and is affiliated with the Energy Biosciences Institute.
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