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Programming and engineering biological networks
Programming and engineering biological networks Jason W Chin Synthetic biology aims to build new functions in living organisms. Recent work has addressed the creation of synthetic epigenetic switches in mammalian cells and synthetic intracellular communication. Fundamentally new, and potentially scaleable, modes of gene regulation have been created that enable expansion of the scope of synthetic circuits. Increasingly sophisticated models of gene regulation that include stochastic effects are beginning to predict the behaviour of small synthetic networks. Overall, these advances suggest that a combination of molecular engineering and systems engineering should allow the creation of living matter capable of performing many useful and novel functions. Addresses MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK Corresponding author: Chin, Jason W ([email protected])
Current Opinion in Structural Biology 2006, 16:551–556 This review comes from a themed issue on Engineering and design Edited by William F DeGrado and Derek N Woolfson Available online 13th July 2006 0959-440X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2006.06.011
Introduction The synthesis of networks of molecules that perform well-defined functions in living organisms is a central aim of the emerging field of synthetic biology [1–4]. Early work in this area has explored the importance of network topology in determining function; toggle switches, oscillators and logic gates (see glossary) have been created in bacteria by the rearrangement of a small set of natural transcription factors and their binding sites into novel networks [5–9]. Here, I review the expansion of this emerging field over the past two years. During this time period, epigenetic switches, first created in bacteria, have been created in eukaryotic cells [10,11]. Progress has also been made on synthetic intercellular networks [12]. Furthermore, it has become increasingly clear that more independently functioning biological components are required in order to make more sophisticated genetic circuits. This realization has spawned efforts to create both novel scaleable and orthogonal modules to regulate expression [13,14,15], and fundamentally new modes of gene regulation [16]. www.sciencedirect.com
Synthetic epigenetic switches Epigenetic toggle switches (see glossary) respond to transient stimuli to elicit changes that persist even once the stimuli are removed. Natural toggle switches coordinate cell fate and cell memory, plant flowering and lysislysogeny decisions. Pioneering work in bacteria has shown that a synthetic toggle switch can be created from two repressor proteins that negatively regulate each other’s expression. The switch can be toggled between two stable expression states by transient chemical or thermal induction [6]. Recently, Fussenegger and colleagues [10] created a synthetic epigenetic toggle switch in mammalian cells using similar principles. This is a significant achievement because gene regulation in eukaryotic cells is more complicated than in prokaryotes and operates using fundamentally different logic [17]. To create the toggle switch, they configured two antibiotic-inducible transcription control systems, PIP ON and E ON, composed of bacterial response regulators fused to the eukaryotic transrepressor domain KRAB, such that they repress each other’s expression (Figure 1). Secreted alkaline phosphatase (SEAP) was co-cistronically expressed with PIP KRAB, providing a measurable output for the switch. They demonstrated that the switch can be repeatedly toggled between high and low expression of SEAP over a period of days in response to transient exposure to the small molecules erythromycin and pristinamycin I. Moreover, once high or low levels of expression of SEAP were set by transient exposure to the appropriate small molecule, the expression levels were maintained for more than three weeks in the absence of the molecule. They went on to show that, when capsules containing CHO cells stably transformed with the genetic toggle switch were intraperitoneally implanted into mice and the mice were injected with either erythromycin or pristinamycin I for 3 days, subsequent SEAP expression levels could be locked. If mammalian toggle switches can be interfaced with cellular networks, as recently demonstrated for bacterial toggle switches [11], it should be possible to track the fate of individual cells and their daughters in multicellular organisms in response to diverse stimuli; this may allow the correlation of cell fate with exposure to stimuli. It will be interesting to see if other types of synthetic circuits that have been created in bacteria can be transferred to eukaryotic organisms.
Synthetic cell–cell communication Cell–cell communication coordinates the behaviour of cells in a population, and is important for defining and maintaining multicellular organism morphology and Current Opinion in Structural Biology 2006, 16:551–556
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Glossary Antisense: nucleic acid molecules that bind a complementary strand of nucleic acid to modify gene expression. Epigenetic toggle switch: a genetic device that allows the state of an organism to be ‘toggled’ by the transient application of a stimulus. In the absence of further stimulus, the new state of the organism is stably inherited, although no genetic change takes place. Logic gate: an arrangement of controlled switches used to calculate operations using Boolean logic in digital circuits. There are three fundamental gates (AND, NOT, OR), from which all others can, in principle, be assembled. Quorum sensing: the communication and coordination of bacteria through signalling molecules.
physiology. Recently, work has begun to create new modes of cell–cell communication in both Escherichia coli and yeast. In E. coli, this work has exploited key components of the natural quorum sensing mechanism (see glossary) of Vibrio fischeri encoded by the Lux operon, including the Lux I and Lux R proteins and the Lux I promoter. Lux I converts metabolites to N-3-oxohexanoyl-L-homoserine (AHL), which freely diffuses between cells; Lux R is a transcriptional activator that is dependent on AHL for activity; and the Lux I promoter is Lux R responsive. Thus, in response to high concentrations of AHL (e.g. at high cell density), transcription of genes placed downstream of the Lux I promoter is activated. In natural quorum sensing, every cell both sends and receives AHL. However, it is possible to dissect the Lux operon and partition its components between distinct populations of cells to create two distinct types of cells: those that synthesize AHL (sender cells) and those that activate gene circuit expression in response to high concentrations of AHL (receiver cells). Weiss and colleagues
[12] created three receiver circuits (Figure 2), each of which is designed to produce a distinct fluorescent protein in response to a different window of AHL concentration. They placed a disk of sender cells in a Petri dish containing a lawn composed of a mixture of all three types of receiver cells. Fluorescent bands of cells formed at discrete distances from the sender cells, corresponding to the AHL response window of each strain. The observed pattern demonstrates that the lawn of genetically distinct, but spatially undifferentiated, cells has been spatially differentiated by the diffusive gradient generated by the manually localized sender cells. Similar principles have been used to create both transient responses to AHL signals [18] and population control circuits [19]. An important challenge will be to show that such synthetic spatial differentiation can be created from genetically identical cells. This might be achieved by creating epigenetic switches that toggle in response to narrow windows of sender molecule concentrations. It may ultimately be possible to extend these approaches to the creation of artificial multicellular morphologies, which may be used for tissue engineering, and imprinting and fabricating materials. However, the more sophisticated and conditional signalling required for such applications will most likely require both epigenetic responses and additional orthogonal modes of cell–cell communication [20,21].
Post-transcriptional control of gene expression using synthetic RNAs Genetic circuits ultimately require control over gene expression at the transcriptional or translational level, but there are currently only a handful of well-characterized independent ways of modulating gene expression
Figure 1
The mammalian genetic toggle switch. (a) PPIRON and PETRON8 are pristinamycin- and erythromycin-dependent promoters, respectively. E KRAB and PIP KRAB are erythromycin- and pristinamycin-dependent repressors fused to KRAB trans-silencing domains, respectively. SEAP is co-cistronically expressed with PIP KRAB and translated via an internal ribosome entry site (IRES). (b) The reversibility of the mammalian toggle switch over ten days. SEAP levels are reset upon exchange of the small-molecule inducers (EM, erythromycin; PI, pristinamycin I). Current Opinion in Structural Biology 2006, 16:551–556
www.sciencedirect.com
Programming and engineering biological networks Chin 553
Figure 2
using natural transcription factors. RNA-based control of gene expression is one promising route to expanding independent control. Inspired by natural riboregulators, Collins and co-workers [13] have created synthetic riboregulators for post-transcriptional gene regulation in E. coli (Figure 3). They appended a nucleotide sequence to the 50 end of a natural transcript. The appended sequence is designed to form a hairpin with the sequence that contains the natural ribosome-binding site (RBS) and repress gene expression by occluding ribosome binding. They produced a second RNA in trans that binds to the cis repressor sequence and thereby releases the natural RBS sequence for translation. Cis repressor sequences base pair with the natural sequence, but are also interspersed with mismatches and provide basal repression in excess of 95%. Translation can be increased by approximately an order of magnitude by the trans-activating RNA and, importantly, in the two cases examined, no transactivation of non-cognate cis repressor sequence was observed. This technology shows promise as a scalable route to gene regulation. Because the method does not, in principle, require a specific promoter or target a specific coding sequence, it should be quite versatile. It might be possible to regulate gene expression from native promoters in the genome in this way, allowing detailed studies of gene regulation networks. In another recent effort at gene regulation using RNA, Bayer and Smolke [14] created antisense (see glossary) agents that incorporate previously described liganddependent riboswitches, and demonstrated that it was possible to repress fluorescent protein expression in a ligand-dependent manner. A remaining challenge for technologies that rely on switching between alternate RNA structures to control gene expression is to exploit the full potential of their dynamic range so that the ‘on’ state exhibits a comparable level of expression to that of an unregulated promoter. It may also be important to further characterize the genome-wide effect of synthetic RNAs on gene expression.
Orthogonal ribosomes The ribosome is a 2.5 MDa molecular machine that synthesizes cellular proteins encoded by mRNA [22]. In bacteria, there is, essentially, only a single type of ribosome, which is essential for protein synthesis and cell survival. We have begun to investigate whether it is possible to diverge the ribosome to create multiple
A pattern-forming circuit. (a) Sender cells broadcast AHL and band detector cells respond to a particular AHL concentration window. Band detector cells contain a feed-forward loop that leads to the observed behaviour by virtue of the differential repression thresholds for green fluorescent protein (GFP) expression in each branch of the loop. Blue stars represent tetracycline, purple circles are AHL. (b) An example of the pattern formed by two different band-forming strains that initially form an undifferentiated lawn of E. coli on an agar plate. www.sciencedirect.com
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Figure 3
Synthetic riboregulator. cr, cis-repressor sequence; P, promoter; taRNA, trans-activating RNA.
orthogonal ribosomes that function independently of the natural ribosome in cells. Natural evolution is believed to have arrived at new molecular and organismal functions by the duplication of existing sets of genes and the alteration of the new copies to perform new functions [23]. We investigated whether we could achieve an analogous effect on a laboratory timescale in two steps, each of which filters a population of cells. In the first step, we created a library of new potential RBSs by creating all possible combinations of nucleotides at each position of the existing RBS [15]. This library was placed upstream of a fusion between the chloramphenicol acetyl transferase gene and a uracil phosphoribosyl transferase gene. The gene fusion is phenotypically silent in the absence of added molecules, but addition of chloramphenicol or 5-fluorouracil (5-FU) enables selection for or against its own expression, respectively. By growing the library of RBS mutants on 5-FU, we were able to select orthogonal mRNA (O-mRNA) sequences that are not translated by the endogenous ribosome. In a second step, the selected O-mRNAs were combined with a library of mutant ribosomes and those mutant ribosomes that specifically translate the selected O-mRNAs were isolated. Finally, we were able to show that selected ribosomes do not significantly translate cellular transcripts and do not significantly alter the cellular growth rate. These ribosomes are therefore orthogonal (Oribosomes). Extensions of this two-step selection approach Current Opinion in Structural Biology 2006, 16:551–556
will allow the engineering of almost any molecular interaction that can be linked to gene expression. The approach provides a powerful route to the creation of new cellular modules with prescribed connections to, and insulation from, other cellular modules. From the two-step selection, we isolated three distinct types of orthogonal pairs (O-ribosomeO-mRNA pairs, Figure 4). The pairs created have several potential applications. As the endogenous ribosome is an essential cellular hub, many mutations are lethal. In contrast, the O-ribosomes are free from the requirement to translate the proteome, and we are currently exploiting this distinction to diverge and expand ribosome function (JW Chin et al., unpublished). This same property, in combination with rRNA mutagenesis methods, has allowed the interrogation of rRNA structure-function relationships on an unprecedented scale. In particular, we recently described functional epitopes in the phylogenetically conserved and structurally defined RNA–RNA ribosome intersubunit interface [24]. Finally, we have shown that several of the pairs are mutually orthogonal and can function simultaneously in a single cell. This property has allowed exploration of a fundamentally new type of logical regulation of function, whereby the translation of genes is controlled by their RBS and by the presence or absence of cognate O-ribosomes [16]. www.sciencedirect.com
Programming and engineering biological networks Chin 555
Figure 4
Orthogonal ribosomes and Boolean logic. (a) The mutual orthogonality of ribosomemRNA pairs that are orthogonal to the endogenous pair is revealed by measuring growth on chloramphenicol (Cm) when the chloramphenicol acetyl transferase gene is placed downstream of cognate and non-cognate RBSs. (b) The network of interactions from (a). Grey lines correspond to weak or unmeasureable interactions, whereas black lines correspond to strong functional interactions. (c) An example of a translational logic gate. Production of ribosomes A AND C is required to reconstitute b-galactosidase from the a and v fragment, as measured by the conversion of fluorescein b-D-galactopyranoside (FDG) to fluorescein (F).
Conclusions In this review, I have focused on recent developments in programming and engineering biological networks to perform new functions. In the future, synthetic epigenetic patterns and morphologies might be addressed, and synthetic circuits may be increasingly integrated with cellular physiology. The increased measurement and understanding of stochastic effects on organism function should make engineering biology more tractable [25,26,27]. Large-scale DNA synthesis [28–30] and error correction methods [31] may make the synthesis of genes encoding the components of any desired circuit routine. This may bring new challenges in both managing the increased metabolic load imposed by larger unnatural networks and ensuring that the molecular components are sufficiently specific to ensure independent function. These exciting challenges will require the concerted effort of molecular www.sciencedirect.com
engineers, systems engineers, biologists and physical scientists.
Acknowledgements JWC is an EMBO Young Investigator.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Chin JW: Modular approaches to expanding the functions of living matter. Nat Chem Biol 2006, 2:304-311.
2.
Sprinzak D, Elowitz MB: Reconstruction of genetic circuits. Nature 2005, 438:443-448.
3.
Endy D: Foundations for engineering biology. Nature 2005, 438:449-453. Current Opinion in Structural Biology 2006, 16:551–556
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4.
Sismour AM, Benner SA: Synthetic biology. Expert Opin Biol Ther 2005, 5:1409-1414.
5.
Elowitz MB, Leibler S: A synthetic oscillatory network of transcriptional regulators. Nature 2000, 403:335-338.
6.
Gardner TS, Cantor CR, Collins JJ: Construction of a genetic toggle switch in Escherichia coli. Nature 2000, 403:339-342.
7.
Guet CC, Elowitz MB, Hsing W, Leibler S: Combinatorial synthesis of genetic networks. Science 2002, 296:1466-1470.
8.
Yokobayashi Y, Weiss R, Arnold FH: Directed evolution of a genetic circuit. Proc Natl Acad Sci USA 2002, 99:16587-16591.
9.
Becskei A, Serrano L: Engineering stability in gene networks by autoregulation. Nature 2000, 405:590-593.
10. Kramer BP, Viretta AU, Daoud-El-Baba M, Aubel D, Weber W, Fussenegger M: An engineered epigenetic transgene switch in mammalian cells. Nat Biotechnol 2004, 22:867-870. The authors report the first synthetic genetic toggle switch in mammalian cells. 11. Kobayashi H, Kaern M, Araki M, Chung K, Gardner TS, Cantor CR, Collins JJ: Programmable cells: interfacing natural and engineered gene networks. Proc Natl Acad Sci USA 2004, 101:8414-8419. A previously described genetic toggle switch is interfaced with the cell’s genetic network to produce unnatural epigenetic changes in response to activation of cellular responses. 12. Basu S, Gerchman Y, Collins CH, Arnold FH, Weiss R: A synthetic multicellular system for programmed pattern formation. Nature 2005, 434:1130-1134. This paper demonstrates spatial pattern formation in response to diffusive gradients. 13. Isaacs FJ, Dwyer DJ, Ding C, Pervouchine DD, Cantor CR, Collins JJ: Engineered riboregulators enable posttranscriptional control of gene expression. Nat Biotechnol 2004, 22:841-847. The clever design of cis and trans riboregulators that might be used as scaleable gene regulation modules is described. 14. Bayer TS, Smolke CD: Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol 2005, 23:337-343. 15. Rackham O, Chin JW: A network of orthogonal ribosome x mRNA pairs. Nat Chem Biol 2005, 1:159-166. The authors report the creation of multiple mutually orthogonal ribosomemRNA pairs for the independent regulation of gene expression. 16. Rackham O, Chin JW: Cellular logic with orthogonal ribosomes. J Am Chem Soc 2005, 127:17584-17585. The orthogonal pairs described in [15] are combined in single cells to produce Boolean logic functions. 17. Struhl K: Fundamentally different logic of gene regulation in eukaryotes and prokaryotes. Cell 1999, 98:1-4.
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18. Basu S, Mehreja R, Thiberge S, Chen MT, Weiss R: Spatiotemporal control of gene expression with pulsegenerating networks. Proc Natl Acad Sci USA 2004, 101:6355-6360. 19. You L, Cox RS III, Weiss R, Arnold FH: Programmed population control by cell-cell communication and regulated killing. Nature 2004, 428:868-871. 20. Chen MT, Weiss R: Artificial cell-cell communication in yeast Saccharomyces cerevisiae using signaling elements from Arabidopsis thaliana. Nat Biotechnol 2005, 23:1551-1555. 21. Bulter T, Lee SG, Wong WW, Fung E, Connor MR, Liao JC: Design of artificial cell-cell communication using gene and metabolic networks. Proc Natl Acad Sci USA 2004, 101:2299-2304. 22. Ramakrishnan V: Ribosome structure and the mechanism of translation. Cell 2002, 108:557-572. 23. Ohno S, Wolf U, Atkin NB: Evolution from fish to mammals by gene duplication. Hereditas 1968, 59:169-187. 24. Rackham O, Wang K, Chin JW: Functional epitopes at the ribosome subunit interface. Nat Chem Biol 2006, 2:254-258. 25. Rosenfeld N, Young JW, Alon U, Swain PS, Elowitz MB: Gene regulation at the single-cell level. Science 2005, 307:1962-1965. 26. Guido NJ, Wang X, Adalsteinsson D, McMillen D, Hasty J, Cantor CR, Elston TC, Collins JJ: A bottom-up approach to gene regulation. Nature 2006, 439:856-860. Models of gene regulation that include stochastic effects allow the behaviour of small gene networks to be predicted. This is a promising approach for building more complex gene circuits de novo from the ‘bottom up’. 27. Cai L, Friedman N, Xie XS: Stochastic protein expression in individual cells at the single molecule level. Nature 2006, 440:358-362. 28. Tian J, Gong H, Sheng N, Zhou X, Gulari E, Gao X, Church G: Accurate multiplex gene synthesis from programmable DNA microchips. Nature 2004, 432:1050-1054. 29. Smith HO, Hutchison CA III, Pfannkoch C, Venter JC: Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides. Proc Natl Acad Sci USA 2003, 100:15440-15445. 30. Kodumal SJ, Patel KG, Reid R, Menzella HG, Welch M, Santi DV: Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster. Proc Natl Acad Sci USA 2004, 101:15573-15578. 31. Binkowski BF, Richmond KE, Kaysen J, Sussman MR, Belshaw PJ: Correcting errors in synthetic DNA through consensus shuffling. Nucleic Acids Res 2005, 33:e55. A clever method with potential for error correction in long synthetic DNAs.
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Current Opinion in Structural Biology 2006, 16:551–556 This review comes from a themed issue on Engineering and design Edited by William F DeGrado and Derek N Woolfson Available online 13th July 2006 0959-440X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2006.06.011
Introduction The synthesis of networks of molecules that perform well-defined functions in living organisms is a central aim of the emerging field of synthetic biology [1–4]. Early work in this area has explored the importance of network topology in determining function; toggle switches, oscillators and logic gates (see glossary) have been created in bacteria by the rearrangement of a small set of natural transcription factors and their binding sites into novel networks [5–9]. Here, I review the expansion of this emerging field over the past two years. During this time period, epigenetic switches, first created in bacteria, have been created in eukaryotic cells [10,11]. Progress has also been made on synthetic intercellular networks [12]. Furthermore, it has become increasingly clear that more independently functioning biological components are required in order to make more sophisticated genetic circuits. This realization has spawned efforts to create both novel scaleable and orthogonal modules to regulate expression [13,14,15], and fundamentally new modes of gene regulation [16]. www.sciencedirect.com
Synthetic epigenetic switches Epigenetic toggle switches (see glossary) respond to transient stimuli to elicit changes that persist even once the stimuli are removed. Natural toggle switches coordinate cell fate and cell memory, plant flowering and lysislysogeny decisions. Pioneering work in bacteria has shown that a synthetic toggle switch can be created from two repressor proteins that negatively regulate each other’s expression. The switch can be toggled between two stable expression states by transient chemical or thermal induction [6]. Recently, Fussenegger and colleagues [10] created a synthetic epigenetic toggle switch in mammalian cells using similar principles. This is a significant achievement because gene regulation in eukaryotic cells is more complicated than in prokaryotes and operates using fundamentally different logic [17]. To create the toggle switch, they configured two antibiotic-inducible transcription control systems, PIP ON and E ON, composed of bacterial response regulators fused to the eukaryotic transrepressor domain KRAB, such that they repress each other’s expression (Figure 1). Secreted alkaline phosphatase (SEAP) was co-cistronically expressed with PIP KRAB, providing a measurable output for the switch. They demonstrated that the switch can be repeatedly toggled between high and low expression of SEAP over a period of days in response to transient exposure to the small molecules erythromycin and pristinamycin I. Moreover, once high or low levels of expression of SEAP were set by transient exposure to the appropriate small molecule, the expression levels were maintained for more than three weeks in the absence of the molecule. They went on to show that, when capsules containing CHO cells stably transformed with the genetic toggle switch were intraperitoneally implanted into mice and the mice were injected with either erythromycin or pristinamycin I for 3 days, subsequent SEAP expression levels could be locked. If mammalian toggle switches can be interfaced with cellular networks, as recently demonstrated for bacterial toggle switches [11], it should be possible to track the fate of individual cells and their daughters in multicellular organisms in response to diverse stimuli; this may allow the correlation of cell fate with exposure to stimuli. It will be interesting to see if other types of synthetic circuits that have been created in bacteria can be transferred to eukaryotic organisms.
Synthetic cell–cell communication Cell–cell communication coordinates the behaviour of cells in a population, and is important for defining and maintaining multicellular organism morphology and Current Opinion in Structural Biology 2006, 16:551–556
552 Engineering and design
Glossary Antisense: nucleic acid molecules that bind a complementary strand of nucleic acid to modify gene expression. Epigenetic toggle switch: a genetic device that allows the state of an organism to be ‘toggled’ by the transient application of a stimulus. In the absence of further stimulus, the new state of the organism is stably inherited, although no genetic change takes place. Logic gate: an arrangement of controlled switches used to calculate operations using Boolean logic in digital circuits. There are three fundamental gates (AND, NOT, OR), from which all others can, in principle, be assembled. Quorum sensing: the communication and coordination of bacteria through signalling molecules.
physiology. Recently, work has begun to create new modes of cell–cell communication in both Escherichia coli and yeast. In E. coli, this work has exploited key components of the natural quorum sensing mechanism (see glossary) of Vibrio fischeri encoded by the Lux operon, including the Lux I and Lux R proteins and the Lux I promoter. Lux I converts metabolites to N-3-oxohexanoyl-L-homoserine (AHL), which freely diffuses between cells; Lux R is a transcriptional activator that is dependent on AHL for activity; and the Lux I promoter is Lux R responsive. Thus, in response to high concentrations of AHL (e.g. at high cell density), transcription of genes placed downstream of the Lux I promoter is activated. In natural quorum sensing, every cell both sends and receives AHL. However, it is possible to dissect the Lux operon and partition its components between distinct populations of cells to create two distinct types of cells: those that synthesize AHL (sender cells) and those that activate gene circuit expression in response to high concentrations of AHL (receiver cells). Weiss and colleagues
[12] created three receiver circuits (Figure 2), each of which is designed to produce a distinct fluorescent protein in response to a different window of AHL concentration. They placed a disk of sender cells in a Petri dish containing a lawn composed of a mixture of all three types of receiver cells. Fluorescent bands of cells formed at discrete distances from the sender cells, corresponding to the AHL response window of each strain. The observed pattern demonstrates that the lawn of genetically distinct, but spatially undifferentiated, cells has been spatially differentiated by the diffusive gradient generated by the manually localized sender cells. Similar principles have been used to create both transient responses to AHL signals [18] and population control circuits [19]. An important challenge will be to show that such synthetic spatial differentiation can be created from genetically identical cells. This might be achieved by creating epigenetic switches that toggle in response to narrow windows of sender molecule concentrations. It may ultimately be possible to extend these approaches to the creation of artificial multicellular morphologies, which may be used for tissue engineering, and imprinting and fabricating materials. However, the more sophisticated and conditional signalling required for such applications will most likely require both epigenetic responses and additional orthogonal modes of cell–cell communication [20,21].
Post-transcriptional control of gene expression using synthetic RNAs Genetic circuits ultimately require control over gene expression at the transcriptional or translational level, but there are currently only a handful of well-characterized independent ways of modulating gene expression
Figure 1
The mammalian genetic toggle switch. (a) PPIRON and PETRON8 are pristinamycin- and erythromycin-dependent promoters, respectively. E KRAB and PIP KRAB are erythromycin- and pristinamycin-dependent repressors fused to KRAB trans-silencing domains, respectively. SEAP is co-cistronically expressed with PIP KRAB and translated via an internal ribosome entry site (IRES). (b) The reversibility of the mammalian toggle switch over ten days. SEAP levels are reset upon exchange of the small-molecule inducers (EM, erythromycin; PI, pristinamycin I). Current Opinion in Structural Biology 2006, 16:551–556
www.sciencedirect.com
Programming and engineering biological networks Chin 553
Figure 2
using natural transcription factors. RNA-based control of gene expression is one promising route to expanding independent control. Inspired by natural riboregulators, Collins and co-workers [13] have created synthetic riboregulators for post-transcriptional gene regulation in E. coli (Figure 3). They appended a nucleotide sequence to the 50 end of a natural transcript. The appended sequence is designed to form a hairpin with the sequence that contains the natural ribosome-binding site (RBS) and repress gene expression by occluding ribosome binding. They produced a second RNA in trans that binds to the cis repressor sequence and thereby releases the natural RBS sequence for translation. Cis repressor sequences base pair with the natural sequence, but are also interspersed with mismatches and provide basal repression in excess of 95%. Translation can be increased by approximately an order of magnitude by the trans-activating RNA and, importantly, in the two cases examined, no transactivation of non-cognate cis repressor sequence was observed. This technology shows promise as a scalable route to gene regulation. Because the method does not, in principle, require a specific promoter or target a specific coding sequence, it should be quite versatile. It might be possible to regulate gene expression from native promoters in the genome in this way, allowing detailed studies of gene regulation networks. In another recent effort at gene regulation using RNA, Bayer and Smolke [14] created antisense (see glossary) agents that incorporate previously described liganddependent riboswitches, and demonstrated that it was possible to repress fluorescent protein expression in a ligand-dependent manner. A remaining challenge for technologies that rely on switching between alternate RNA structures to control gene expression is to exploit the full potential of their dynamic range so that the ‘on’ state exhibits a comparable level of expression to that of an unregulated promoter. It may also be important to further characterize the genome-wide effect of synthetic RNAs on gene expression.
Orthogonal ribosomes The ribosome is a 2.5 MDa molecular machine that synthesizes cellular proteins encoded by mRNA [22]. In bacteria, there is, essentially, only a single type of ribosome, which is essential for protein synthesis and cell survival. We have begun to investigate whether it is possible to diverge the ribosome to create multiple
A pattern-forming circuit. (a) Sender cells broadcast AHL and band detector cells respond to a particular AHL concentration window. Band detector cells contain a feed-forward loop that leads to the observed behaviour by virtue of the differential repression thresholds for green fluorescent protein (GFP) expression in each branch of the loop. Blue stars represent tetracycline, purple circles are AHL. (b) An example of the pattern formed by two different band-forming strains that initially form an undifferentiated lawn of E. coli on an agar plate. www.sciencedirect.com
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554 Engineering and design
Figure 3
Synthetic riboregulator. cr, cis-repressor sequence; P, promoter; taRNA, trans-activating RNA.
orthogonal ribosomes that function independently of the natural ribosome in cells. Natural evolution is believed to have arrived at new molecular and organismal functions by the duplication of existing sets of genes and the alteration of the new copies to perform new functions [23]. We investigated whether we could achieve an analogous effect on a laboratory timescale in two steps, each of which filters a population of cells. In the first step, we created a library of new potential RBSs by creating all possible combinations of nucleotides at each position of the existing RBS [15]. This library was placed upstream of a fusion between the chloramphenicol acetyl transferase gene and a uracil phosphoribosyl transferase gene. The gene fusion is phenotypically silent in the absence of added molecules, but addition of chloramphenicol or 5-fluorouracil (5-FU) enables selection for or against its own expression, respectively. By growing the library of RBS mutants on 5-FU, we were able to select orthogonal mRNA (O-mRNA) sequences that are not translated by the endogenous ribosome. In a second step, the selected O-mRNAs were combined with a library of mutant ribosomes and those mutant ribosomes that specifically translate the selected O-mRNAs were isolated. Finally, we were able to show that selected ribosomes do not significantly translate cellular transcripts and do not significantly alter the cellular growth rate. These ribosomes are therefore orthogonal (Oribosomes). Extensions of this two-step selection approach Current Opinion in Structural Biology 2006, 16:551–556
will allow the engineering of almost any molecular interaction that can be linked to gene expression. The approach provides a powerful route to the creation of new cellular modules with prescribed connections to, and insulation from, other cellular modules. From the two-step selection, we isolated three distinct types of orthogonal pairs (O-ribosomeO-mRNA pairs, Figure 4). The pairs created have several potential applications. As the endogenous ribosome is an essential cellular hub, many mutations are lethal. In contrast, the O-ribosomes are free from the requirement to translate the proteome, and we are currently exploiting this distinction to diverge and expand ribosome function (JW Chin et al., unpublished). This same property, in combination with rRNA mutagenesis methods, has allowed the interrogation of rRNA structure-function relationships on an unprecedented scale. In particular, we recently described functional epitopes in the phylogenetically conserved and structurally defined RNA–RNA ribosome intersubunit interface [24]. Finally, we have shown that several of the pairs are mutually orthogonal and can function simultaneously in a single cell. This property has allowed exploration of a fundamentally new type of logical regulation of function, whereby the translation of genes is controlled by their RBS and by the presence or absence of cognate O-ribosomes [16]. www.sciencedirect.com
Programming and engineering biological networks Chin 555
Figure 4
Orthogonal ribosomes and Boolean logic. (a) The mutual orthogonality of ribosomemRNA pairs that are orthogonal to the endogenous pair is revealed by measuring growth on chloramphenicol (Cm) when the chloramphenicol acetyl transferase gene is placed downstream of cognate and non-cognate RBSs. (b) The network of interactions from (a). Grey lines correspond to weak or unmeasureable interactions, whereas black lines correspond to strong functional interactions. (c) An example of a translational logic gate. Production of ribosomes A AND C is required to reconstitute b-galactosidase from the a and v fragment, as measured by the conversion of fluorescein b-D-galactopyranoside (FDG) to fluorescein (F).
Conclusions In this review, I have focused on recent developments in programming and engineering biological networks to perform new functions. In the future, synthetic epigenetic patterns and morphologies might be addressed, and synthetic circuits may be increasingly integrated with cellular physiology. The increased measurement and understanding of stochastic effects on organism function should make engineering biology more tractable [25,26,27]. Large-scale DNA synthesis [28–30] and error correction methods [31] may make the synthesis of genes encoding the components of any desired circuit routine. This may bring new challenges in both managing the increased metabolic load imposed by larger unnatural networks and ensuring that the molecular components are sufficiently specific to ensure independent function. These exciting challenges will require the concerted effort of molecular www.sciencedirect.com
engineers, systems engineers, biologists and physical scientists.
Acknowledgements JWC is an EMBO Young Investigator.
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