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regulation of transcription by unnatural amino acids

Chang C Liu1,2, Lei Qi1, Charles Yanofsky3 & Adam P Arkin1,4,5
Small-molecule regulation of gene expression is intrinsic to cellular function and indispensable to the construction of new biological sensing, control and expression systems1,2. However, there are currently only a handful of strategies for engineering such regulatory components and fewer still that can give rise to an arbitrarily large set of inducible systems whose members respond to different small molecules, display uniformity and modularity in their mechanisms of regulation, and combine to actuate universal logics38. Here we present an approach for small-molecule regulation of transcription based on the combination of cis-regulatory leader-peptide elements with genetically encoded unnatural amino acids (amino acids that have been artificially added to the genetic code). In our system, any genetically encoded unnatural amino acid (UAA) can be used as a small-molecule attenuator or activator of gene transcription, and the logics intrinsic to the network defined by expanded genetic codes can be actuated. Cis-regulatory leader-peptide elements serve as 5 transcriptional regulation units in which the successful translation of an embedded leader peptide actuates a change in the transcription of the regulated downstream gene(s). We adopted two such elements for this study. The first is the trp operons regulatory region whose wild-type function is the transcriptional attenuation of downstream genes in the presence of tryptophan (Supplementary Fig. 1a)9. In this process, a low concentration of tryptophan (and more directly, tryptophancharged tRNATrp) results in ribosomal stalling at either of two tryptophan codons during translation of the leader-peptide coding region. Stalling interferes with the formation of structure 1:2 and instead triggers structure 2:3, which in turn prevents the formation of the transcriptional terminator structure 3:4 and results in continuation of transcription into the downstream genes. In contrast, when exogenous tryptophan is present at high concentrations and the cellular tRNATrp is mostly charged, the ribosome does not stall at stem-loop 1:2. Stem-loop 1:2 remains intact and the transcriptional terminator 3:4 properly forms, abrogating transcription of the downstream genes. The second cis-regulatory mechanism that we adopted is that of the tna operon (Supplementary Fig. 1b)10. Here, successful translation of the leader-peptide transcript segment results in the inhibition of ribosomal release when sufficient free tryptophan is present. As the leader-peptide coding sequence is followed by a boxA site, a Rho termination factor binding site (rut) and a noncoding section where Rho factordependent transcriptional termination can occur, prolonged ribosomal stalling in this region prevents Rho factor binding and termination. The downstream genes are therefore transcribed. In contrast, if excess tryptophan is unavailable or if the leader peptide is not fully translated, the translating ribosome is not able to block the rut site and Rho factormediated termination takes place. The downstream genes are therefore not transcribed. In summary, we adopted two cis-regulatory elements that complement each other in that one attenuates transcription upon the proper synthesis of the leader peptide and the other activates transcription upon the proper synthesis of the leader peptide. We hypothesized that if we introduced blank codons (codons that do not encode a natural proteinogenic amino acid and include nonsense and frameshift codons) in the right positions in these leaderpeptide coding regions, we would prevent their proper translation and lock transcription of any downstream gene(s) in one state: for the trp regulatory element, transcription of the downstream gene(s) would be active; for the tna regulatory element, transcription of the downstream gene(s) would be inactive. If we then introduced tRNAs that could decode these blank codons (codonBLs) and the corresponding aminoacyl-tRNA synthetases (aaRSs) that could charge the tRNAs with UAAs11, we could induce the successful translation of the leader peptides upon the addition of the proper UAA. This would then effect a change in the transcription of the downstream gene(s): for the trp regulatory element, transcription of the downstream gene(s) would be inhibited; for the tna regulatory element, transcription of the downstream gene(s) would be activated. The result would be two gene expression control switches: a transcriptional OFF switch in which the addition of a specific UAA inhibits transcription of desired downstream gene(s) and a transcriptional ON switch in which the addition of a specific UAA activates transcription of desired downstream gene(s) (Supplementary Fig. 2). There would be several important advantages to these systems (Fig. 1). First, because transcriptional control elements act in cis, they could in theory be placed (either as single units or in tandem) upstream of any gene (or many genes) to effect independent expression control by specific UAAs. Second, because the small-molecule inducers of the OFF and ON switches would rely on components engineered for expanded genetic codesthese are orthogonal aaRS/tRNA pairs specific for various UAAs, and ~60 such systems have been reported for Escherichia coli as a result of standardized methods for genetic code expansion11the number of UAA-induced switches that could readily

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1Department of Bioengineering, University of California at Berkeley, Berkeley, California, USA. 2Miller Institute for Basic Research in Science, Berkeley, California, USA. 3Department of Biological Sciences, Stanford University, Stanford, California, USA. 4Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. 5QB3: California Institute for Quantitative Biological Research, University of California at Berkeley, Berkeley, California, USA. Correspondence should be addressed to C.C.L. (ccliu@berkeley.edu) or A.P.A. (aparkin@lbl.gov).

Received 23 July 2010; accepted 1 December 2010; published online 16 January 2011; doi:10.1038/nbt.1741

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Control, sensing

UAAs

- A large set of synthetic small-molecule inducers - Sensing for biologically relevant UAAs

aaRSs
Logics

tRNAs

- Molecular recognition patterns define logics - Homogeneous and modular components - Systematic methods for new aaRSs and tRNAs

CodonBLs

Integration and actuation

Leader peptide

Regulated genes

Leader-peptide elements

- In cis transcriptional regulation - Combined regulatory units for higher-order control - Regulation of gene cassettes - Separate regulators for separate genes possible

2011 Nature America, Inc. All rights reserved.

Figure 1 A representative molecular recognition network of expanded genetic codes, its integration with cis-regulatory leader-peptide elements and some possible uses of this platform. Solid lines signify productive recognition between the connected components. The UAA-specific aaRS/tRNA pairs represented in this figure are the result of efforts to expand the genetic codes of organisms. This has lead to the successful encoding of ~70 UAAs that encompass a wide range of chemical structures. As we do not discuss the expanded genetic code field in detail, please refer to reference 11.

be made is large and ever-expanding. Third, because each distinct UAA-induced switch would rely on the same basic mechanism of synthetase recognition, aminoacylation, leader-peptide translation and subsequent transcriptional control, one could expect homogeneous responses across the large set of possible derived switches. Fourth, because the network of molecular recognition events defining expanded genetic codes is modular and rich, there are many components that could be systematically combined to implement logics in these switches. Finally, because UAAs can be intermediates in several important metabolic pathways12,13, the switches could be used as sensors that actuate a change in gene expression in conjunction with the activities of certain natural or engineered cellular processes. For these reasons and others, we were interested in realizing such UAAcontrolled cis-regulatory transcriptional switches. Here, we describe their design, characterization, extension to multiple UAAs and logical integration of multiple UAA inputs. To engineer a UAA-induced transcriptional OFF switch, we replaced the second tryptophan codon in the trp operons leader peptide with a codonBL, specifically the amber nonsense codon UAG. DNA corresponding to this mutant cis-regulatory region was then cloned upstream of the reporter gene encoding the superfolder green fluorescent protein (GFP)14 and inserted into a low-copy pSC101derived vector under the control of a constitutive promoter to yield plasmid pCCL-006. E. coli BL21(DE3) cells were then transformed with pCCL-006 and grown in selective 2YT rich media. (Rich media, which contains excess tryptophan, was used throughout this study, as the presence of tryptophan is required for the function of both our OFF switch and the ON switch discussed below.) Cells containing only pCCL-006 displayed high fluorescence, suggesting that the UAG codon placed in the cis-regulatory region sufficiently induces ribosomal stalling in stem-loop 1:2 and formation of the 2:3 structure, allowing transcription of the downstream reporter gene GFP (Fig. 2a). This activity is consistent with previous studies in which the mutation of a trp operonsensing codon to the opal nonsense codon resulted in increased downstream transcription15. Next, we tested whether efficient suppression of the UAG codon would trigger transcriptional termination. To do this, we cotransformed E. coli cells with plasmids pCCL-006 and pEVOL-Tyr. pEVOL-Tyr encodes an
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engineered orthogonal tyrosyl-tRNA synthetase (TyrRS)/tRNACUA pair, derived from the Methanocaldococcus jannaschii TyrRS/tRNATyr pair, that allows the specific incorporation of tyrosine in response to the UAG codon in E. coli16. We therefore expected a decrease in cellular fluorescence as a result of translational readthrough of the UAG codon and subsequent transcriptional termination by the 3:4 terminator structure. This is what we observed. Finally, to obtain a UAA-controlled OFF switch, E. coli cells were cotransformed with plasmids pCCL-006 and pEVOL-pAcF (Fig. 2a). As pEVOL-pAcF encodes an engineered orthogonal aaRS/tRNACUA pair specific for the UAA para-acetylphenylalanine (AcF)16,17, we expected ribosomal stalling and transcriptional continuation in the absence of AcF and ribosomal readthrough and transcriptional termination in the presence of AcF. We indeed observed a sixfold decrease in fluorescence upon the addition of AcF (Fig. 2a). As this effect is specific to cells containing both pCCL-006 and pEVOLpAcF, we have achieved a UAA-induced transcriptional OFF switch. Furthermore, as attenuation shows a standard concentration dependence on AcF (Supplementary Fig. 3a) and a unimodal population response (Supplementary Fig. 4a), this OFF switch is titratable and stable across individual cells. We note that the observed sixfold attenuation corresponds to the maximal response allowed by the wild-type trp cis-regulatory leaderpeptide element. This is because in the absence of ribosomal stalling in stem-loop 1:2, the ribosome proceeds to the leader peptides natural stop codon (located at the top of stem-loop 1:2) and acts to stabilize structure 1:2 and the resulting terminator 3:4 (Supplementary Fig. 1a). However, ~24% of the time, the ribosome releases from this location before the RNA polymerase transcribes 3:4; in these cases, the subsequently transcribed RNA forms the 3:4 structure (resulting in termination) or the 2:3 structure (resulting in continued transcription) with approximately equal likelihood, thus yielding an observed 15% basal level of transcription18. Although it may be possible to reduce this basal expression level by engineering terminator strength or tuning the preference for the relevant RNA structures, the basal level, compared to the 90% transcriptional efficiency observed when ribosomal stalling induces structure 2:3 (ref. 15), constitutes a sixfold dynamic range for the wild-type trp leader-peptide regulatory
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Figure 2 Behavior of UAA-controlled transcriptional switches. Fluorescence of cells grown in the presence or absence of para-acetylphenylalanine (5 mM) is shown. GFP expression is shown in background-subtracted relative fluorescence units (RFUs) normalized to optical density (OD); excitation at 485 nm, emission at 510 nm. Background autofluorescence was determined by measuring the RFU/OD of similarly grown cells containing plasmid pCCL-000, a control plasmid that does not encode GFP. Experiments were conducted in triplicate (error bars are s.d.) on the same day. Data were collected using a fluorescence plate reader (Online Methods). (a) Transcriptional OFF switch. (b) Transcriptional ON switch.

region. Therefore, we conclude that addition of AcF in our OFF switch produced the wild-type level of termination. To engineer a UAA-induced transcriptional ON switch, we mutated the tna leader-peptide element to include the UAG codon in its leaderpeptide coding region. First, the wild-type tna cis-regulatory leaderpeptide element, under the control of a constitutive promoter, was placed upstream of a reporter gene encoding superfolder GFP. Next, we added a TAT or a TAG sequence at five locations (replacements at positions 2, 3 and 4; and insertions after position 8 and position 10) in the leader-peptide coding region, taking care not to disrupt predicted RNA secondary structures, and compared the regulatory activities of the resulting mutants to that of the wild-type peptide sequence. (We chose TAT for the positive control mutants because it is similar in sequence to TAG and specifies tyrosine, which is similar in structure to the UAA AcF.) As expected, the TAG variants allowed little GFP expression whereas in four of the five test cases, the corresponding TAT variants enabled considerable GFP expression ranging from 2570% of wild-type levels when grown in 2YT rich media
Figure 3 Control of transcriptional OFF and transcriptional ON switches with multiple UAAs. GFP expression is shown in background-subtracted relative fluorescence units (RFUs) normalized to optical density (OD); excitation at 485 nm, emission at 510 nm. Background autofluorescence was determined by measuring the RFU/OD of similarly grown cells containing plasmid pCCL-000, a control plasmid that does not encode GFP. Experiments were conducted in triplicate (error bars are s.d.) on the same day. Data were collected using a fluorescence plate reader (see Online Methods). (a,b) Fluorescence of cells grown in the presence or absence of para-azidophenylalanine (1 mM), 4-boronophenylalanine (1 mM) or paraiodophenylalanine (1 mM). (c) NOR behavior: cells containing pCCL-006 and pEVOL-Dual-pAcF/pAzF grown in the absence of UAAs, presence of paraacetylphenylalanine (5 mM), presence of para-azidophenylalanine (5 mM), or presence of para-acetylphenylalanine (2.5 mM) and para-azidophenylalanine (2.5 mM). See Supplementary Figure 6a for a mechanistic schematic. (d) OR behavior: cells containing pCCL-016 and pEVOL-Dual-pAcF/pAzF grown in the absence of UAAs, presence of para-acetylphenylalanine (5 mM), presence of para-azidophenylalanine (5 mM), or presence of para-acetylphenylalanine (2.5 mM) and para-azidophenylalanine (2.5 mM). See Supplementary Figure 6b for a mechanistic schematic.

(Supplementary Fig. 5). We therefore reasoned that these four sites were likely suitable for UAA induction of downstream transcription. One of the mutant pairs, contained in plasmids pCCL-015 (TAT insertion after position 10) and pCCL-016 (TAG insertion after position 10), was chosen for characterization of ON switch activity. When E. coli Top10 cells were transformed with pCCL-015 and grown in selective 2YT rich media, high fluorescence was observed, consistent with proper translation of the leader peptide and subsequent inhibition of Rho factordependent translational termination by ribosomal stalling over the natural stop codon and adjacent rut sites (Fig. 2b). Similarly, high fluorescence was observed when E. coli cells were cotransformed with pCCL-016 and pEVOL-Tyr and grown in rich media, because the orthogonal TyrRS/tRNACUA pair encoded by pEVOL-Tyr allows the translational incorporation of tyrosine in response to the UAG codon in pCCL-016s leader peptide16. To obtain a UAA-controlled ON switch, we then cotransformed E. coli cells with pCCL-016 and pEVOL-pAcF. Given that pEVOL-pAcF encodes an orthogonal aaRS/tRNACUA pair specific for the UAA AcF16,17, we expected proper translation of the leader peptide (and subsequent transcription of the reporter GFP) only in the presence of AcF. Indeed, we observed a 25-fold increase in fluorescence upon the addition of AcF (Fig. 2b). As this effect is specific to cells containing both pCCL-016 and pEVOL-pAcF, we have achieved a UAA-induced transcriptional ON switch. Furthermore, as the AcF-induced increase in aggregate fluorescence displays a standard concentration dependence (Supplementary Fig. 3b) and represents a unimodal population response (Supplementary Fig. 4b), this ON switch behavior is titratable and stable across cells. In our OFF and ON transcriptional switches, translation of the leader peptides results in the covalent incorporation of UAAs. The potential structural perturbation introduced by the UAAs, however, should not affect our leader peptides regulatory roles. This is because in the case of the trp operon leader-peptide element, attenuation is dictated by the kinetics of translation and not by the exact composition of the nascent peptide9; and in the case of the tna operon leaderpeptide element, the crucial step of prolonged ribosomal stalling over the rut site is triggered by binding of the leader peptides 12 C-terminal amino acids and not by interactions with the earlier positions in the peptide where our UAAs are incorporated19. Therefore, the function

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of our OFF and ON transcriptional switches should rely only on the proper recognition of the inducer UAA by its corresponding aaRS, and induction of our switches can be extended to any genetically encodable UAA. To test this notion, we cotransformed E. coli cells with pCCL-006 along with pEVOL-pAzF (this encodes an orthogonal aaRS/tRNACUA pair specific for the UAA para-azidophenylalanine), pEVOLBoF (this encodes an orthogonal aaRS/tRNACUA pair specific for the UAA 4-boronophenylalanine) or pEVOL-IF (this encodes an orthogonal aaRS/tRNACUA pair specific for the UAA paraiodophenylalanine)16, and attenuation of GFP fluorescence upon addition of para-azidophenylalanine, 4-boronophenylalanine or para-iodophenylalanine, respectively, was measured. UAA-induced OFF switch behavior was uniformly observed in all cases (Fig. 3a). Analogously, cotransformation of cells with pCCL-016 along with pEVOL-pAzF, pEVOL-BoF or pEVOL-IF resulted in ON switch activities that were uniformly induced by their respective UAAs (Fig. 3b). Therefore, our OFF and ON transcriptional switches are general to all four genetically encoded UAA systems tested (the three described here plus the one encoded by pEVOL-pAcF described earlier). As all genetically encoded UAA systems use the same fundamental mechanism for UAA incorporation, our OFF and ON transcriptional switches should be general to all genetically encoded UAAs. The adaptation of cis-regulatory leader-peptide elements for control by UAAs not only allows this extension of transcriptional regulation to numerous different UAA inducers, but also facilitates the actuation of logic operations implied by the rich network of UAAs, aaRSs, tRNAs and codonBLs defined by expanded genetic codes (Fig. 1). (For example, one can enumerate a set of aaRSs whose members have different UAA specificities but all recognize the same tRNA; one can also assemble a list of aaRSs, some of which only recognize one tRNA and some of which only recognize a second tRNA; one can find examples of aaRSs that recognize more than one UAA or examples of aaRSs known to recognize only one UAA; and one can often change the anticodon of tRNAs to specifically decode any of several possible codonBLs such as nonsense or frameshift codons.) As a result, logics involving UAA inputs can be made available for gene expression control. To demonstrate this feature, we created a NOR gate and an OR gate, each of which uses two UAA inputs. We took advantage of the fact that the aaRS specific for AcF and the aaRS specific for paraazidophenylalanine (AzF) both recognize the same tRNACUA (derived from the M. jannaschii tRNATyr)17,20. Therefore, when pCCL-006 was cotransformed with a plasmid expressing both aaRSswe cloned such a plasmid, pEVOL-Dual-pAcF/pAzFwe expected transcriptional attenuation in the presence of AcF, AzF or both (NOR behavior; Supplementary Fig. 6a); likewise, when pCCL-016 was cotransformed with pEVOL-Dual-pAcF/pAzF, we expected transcriptional activation in the presence of AcF, AzF or both (OR behavior; Supplementary Fig. 6b). These behaviors were indeed obtained (Fig. 3c,d). In the absence of both UAAs, the NOR gate (pCCL-006 + pEVOL-Dual-pAcF/pAzF) was ON and the OR gate (pCCL-016 + pEVOL-Dual-pAcF/pAzF) was OFF; whereas in the presence of either or both of the UAAs, the NOR gate was OFF and the OR gate was ON. Considering that the whole-genome engineering and expanded genetic code fields are rapidly developing more codonBLs and mutually orthogonal aaRS/tRNA pairs to simultaneously specify two or more UAAs (Supplementary Discussion)2125, the analogous multiinput NAND and AND gates should become accessible simply by using different codonBLs in our leader peptides as our switches are cis-regulatory transcriptional units.
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Future work using our platform should lead to the realization of higher-order UAA-induced gene regulation (e.g., a band-pass filter or an all-or-none response), independent but homogeneous control of multiple genes with different UAAs specified by different blank codons (this should facilitate the creation of sophisticated gene circuits or switchboards in a more predictable manner)26,27, sensing or dynamic gene regulation in important metabolic engineering efforts involving UAA intermediates or products (e.g., nonribosomal peptide synthesis, biosynthesis of industrial nonnatural chiral amino acid precursors for drug synthesis, etc.)12,13 and the creation of synthetic cell-cell communication systems using metabolic pathways that yield cell-permeable UAAs. In addition, our strategy can be extended to a wide range of hosts, including yeast and mammalian cells, as cisregulatory leader-peptide elements are widespread (e.g., PheA and ilvB transcriptional regulation in bacteria; arg-2 and CPA1 translational regulation in fungi; and 2-adrenergic receptor, RAR-2 and AdoMetDC translational regulation in mammals)2830 and organisms that have been subject to genetic code expansion are many (e.g., E. coli, Mycobacteria, Saccharomyces cerevisiae, Pichia pastoris and several rodent and human cell lines and primary cells)11. We are actively pursuing these possibilities with the expectation that our strategy for transcriptional regulation with UAA-based translational controland more generally the development of scalable, homogeneous, composable and connectable components26will form a basis for predictable biological design. MetHodS Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturebiotechnology/.
Note: Supplementary information is available on the Nature Biotechnology website. ACknowLedgments We thank P. Schultz (The Scripps Research Institute) for thoughtful comments and the gift of the pEVOL plasmids. We thank J. Lucks for helpful discussions and advice. This work was funded by the National Science Foundation as part of the Synthetic Biology Engineering Research Center (A.P.A.) and the Miller Institute for Basic Scientific Research (C.C.L.). AUtHoR ContRIBUtIons C.C.L. conceived of the study and C.Y. and A.P.A. advised. All authors were involved in designing the experiments. C.C.L. and L.Q. performed the experiments and interpreted the data. C.C.L. and A.P.A. wrote the manuscript. All authors discussed results and commented on the manuscript. ComPetIng FInAnCIAL InteRests The authors declare no competing financial interests.
Published online at http://www.nature.com/naturebiotechnology/. reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/.

1. Roth, A. & Breaker, R.R. The structural and functional diversity of metabolitebinding riboswitches. Annu. Rev. Biochem. 78, 305334 (2009). 2. Keasling, J.D. Synthetic biology for synthetic chemistry. ACS Chem. Biol. 3, 6476 (2008). 3. Isaacs, F.J. et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat. Biotechnol. 22, 841847 (2004). 4. Callura, J.M. et al. Tracking, tuning, and terminating microbial physiology using synthetic riboregulators. Proc. Natl. Acad. Sci. USA 107, 1589815903 (2010). 5. Yen, L. et al. Exogenous control of mammalian gene expression through modulation of RNA self-cleavage. Nature 431, 471476 (2004). 6. Win, M.N. & Smolke, C.D. Higher-order cellular information processing with synthetic RNA devices. Science 322, 456460 (2008). 7. Sharma, V., Nomura, Y. & Yokobayashi, Y. Engineering complex riboswitch regulation by dual genetic selection. J. Am. Chem. Soc. 130, 1631016315 (2008). 8. Anderson, J.C., Voigt, C.A. & Arkin, A.P. Environmental signal integration by a modular AND gate. Mol. Syst. Biol. 3, 133 (2007).

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9. Kolter, R. & Yanofsky, C. Attenuation in amino acid biosynthetic operons. Annu. Rev. Genet. 16, 113134 (1982). 10. Gong, F. & Yanofsky, C. Instruction of translating ribosome by nascent peptide. Science 297, 18641867 (2002). 11. Liu, C.C. & Schultz, P.G. Adding new chemistries to the genetic code. Annu. Rev. Biochem. 79, 413444 (2010). 12. Finking, R. & Marahiel, M.A. Biosynthesis of nonribosomal peptides. Annu. Rev. Microbiol. 58, 453488 (2004). 13. Zhang, K., Li, H., Cho, K.M. & Liao, J.C. Expanding metabolism for total biosynthesis of the nonnatural amino acid L-homoalanine. Proc. Natl. Acad. Sci. USA 107, 62346239 (2010). 14. Pdelacq, J.-D. et al. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 7988 (2006). 15. Landick, R., Yanofsky, C., Choo, K. & Phun, L. Replacement of the Escherichia coli trp operon attenuation control codons alters operon expression. J. Mol. Biol. 216, 2537 (1990). 16. Young, T.S., Ahmad, I., Yin, J.A. & Schultz, P.G. An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395, 361374 (2010). 17. Wang, L., Zhang, Z., Brock, A. & Schultz, P.G. Addition of the keto functional group to the genetic code of Escherichia coli. Proc. Natl. Acad. Sci. USA 100, 5661 (2003). 18. Roesser, J.R., Nakamura, Y. & Yanofsky, C. Regulation of basal level expression of the tryptophan operon of Escherichia coli. J. Biol. Chem. 264, 1228412288 (1989). 19. Seidelt, B. et al. Structural insight into nascent polypeptide chain-mediated translational stalling. Science 326, 14121415 (2009). 20. Chin, J.W. et al. Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. J. Am. Chem. Soc. 124, 90269027 (2002). 21. Wang, H.H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894898 (2009). 22. Neumann, H., Slusarczyk, A.L. & Chin, J.W. De novo generation of mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. J. Am. Chem. Soc. 132, 21422144 (2010). 23. Anderson, J.C. et al. An expanded genetic code with a functional quadruplet codon. Proc. Natl. Acad. Sci. USA 101, 75667571 (2004). 24. Neumann, H. et al. Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Nature 464, 441444 (2010). 25. Wan, W. et al. A facile system for genetic incorporation of two different noncanonical amino acids into one protein in Escherichia coli. Angew. Chem. Int. Ed. 49, 32113214 (2010). 26. Lucks, J.B., Qi, L., Whitaker, W.R. & Arkin, A.P. Toward scalable parts families for predictable design of biological circuits. Curr. Opin. Microbiol. 11, 567573 (2008). 27. Lu, T.K., Khalil, A.S. & Collins, J.J. Next-generation synthetic gene networks. Nat. Biotechnol. 27, 11391150 (2009). 28. Naville, M. & Gautheret, D. Transcription attenuation in bacteria: theme and variations. Brief. Funct. Genomics Proteomics 8, 482492 (2009). 29. Lovett, P.S. & Rogers, E.J. Ribosome regulation by the nascent peptide. Microbiol. Rev. 60, 366385 (1996). 30. Morris, D.R. & Geballe, A.P. Upstream open reading frames as regulators of mRNA translation. Mol. Cell. Biol. 20, 86358642 (2000).

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Plasmid construction. We started from a cloning plasmid, pCCL-000, containing a pSC101 origin of replication and an ampicillin-resistance marker. Next, we amplified a cassette from pAPA1272 (Arkin Laboratory plasmid collection) containing a reporter gene encoding superfolder GFP under the control of promoter J23119(SpeI). Promoter J23119(SpeI) is identical to J23119 except for the replacement of the final six nucleotides with a SpeI site. (J23119 is a strong constitutive promoter whose sequence can be found in the Registry of Standard Biological Parts; http://partsregistry.org/Main_Page.) This cassette was inserted between the AatII and AvrII sites of pCCL-000 (Supplementary List of Plasmids) to yield pCCL-001, which was subsequently modified to contain adjacent SpeI and BamHI sites between promoter J23119(SpeI) and the reporter superfolder GFP. The resulting plasmid, pCCL-002 (Supplementary Sequences), allows for the straightforward insertion of regulatory elements between the SpeI and BamHI sites. To obtain plasmid pCCL-006, a 366-bp DNA fragment (Supplementary Sequences) containing the E. coli trp operon regulatory leader-peptide element with a TAG at position 11 of the leader-peptide open reading frame was custom synthesized (Integrated DNA Technologies). This fragment, flanked by NheI and BamHI sites, was then inserted into pCCL-002 using the SpeI and BamHI sites (NheI and SpeI have compatible ends) as a transcriptional fusion to the superfolder GFP preceded by its own ribosomal binding site. The resulting plasmid, pCCL-006, therefore contains the superfolder GFP gene under the control of a variant trp regulatory leader-peptide element, driven by a variant J23119(SpeI) promoter. To obtain plasmids containing the tna regulatory leader-peptide element and its desired mutants, a DNA fragment corresponding to the upstream regulatory region of the E. coli tna operon was custom synthesized (Integrated DNA Technologies). This 349-bp regulatory region (Supplementary Sequences), which contains an embedded leader-peptide open reading frame (tnaC) and ends with the first ten codons of the tnaA gene (this is the natural gene that is under transcriptional control by the leader-peptide element), was inserted into pCCL-002 as a direct fusion to the coding region of the superfolder GFP gene. The resulting plasmid, pCCL-008, therefore contains the superfolder GFP gene under the control of the tna regulatory leader-peptide element, driven by a J23119(SpeI) promoter. Using Phusion Site-Directed Mutagenesis (New England Biolabs) of pCCL-008, we then generated plasmids pCCL-009 and pCCL-010 (mutation to TAT and TAG, respectively, at tnaC leader peptide position 2), plasmids pCCL-011 and pCCL-012 (mutation to TAT and TAG, respectively, at tnaC leader peptide position 3), plasmids pCCL-013 and pCCL-014 (mutation to TAT and TAG, respectively, at tnaC leader peptide position 4), plasmids pCCL-015 and pCCL-016 (insertion of TAT and TAG, respectively, after tnaC leader peptide position 10) and plasmids pCCL-017 and pCCL-018 (insertion of TAT and TAG, respectively, after tnaC leader peptide position 8). To obtain plasmid pEVOL-Dual-pAcF/pAzF, we amplified a fragment from pEVOL-pAzF using primers 5-GCGTAGAGCTCAAGAAACCAATTGTCCA TAT and 5-CCGGGAGCTCACAAACAAGG. The resulting product, which encodes an aaRS specific for para-azidophenylalanine, was then digested with SacI and inserted into the SacI site of pEVOL-pAcF to yield pEVOL-DualpAcF/pAzF. All plasmid propagation steps during cloning were done in Top10 cells (Invitrogen). Cell culture. To characterize OFF switch and NOR gate behavior, we transformed BL21(DE3) cells (Novagen) with plasmid pCCL-000 or pCCL-006; we also cotransformed BL21(DE3) cells with pCCL-006 along with pEVOLTyr, pEVOL-pAcF, pEVOL-pAzF, pEVOL-BoF, pEVOL-IF or pEVOL-DualpAcF/pAzF. (pEVOL plasmids were the gift of P. Schultz.) The cells were then plated on solid Luria-Bertani (LB) media (Difco) supplemented with ampicillin (100 g/ml) and, in the case of cells also containing pEVOL plasmids, chloramphenicol (30 g/ml). After overnight incubation at 37 C, a colony for each plasmid combination was used to inoculate liquid 2YT media (5 ml, Teknova) containing ampicillin (100 g/ml) and chloramphenicol (30 g/ml) when appropriate. These cultures were shaken overnight at 200 r.p.m. at 37 C. We used 5 l of each overnight culture to inoculate 500 l of 2YT containing 0.2% l-arabinose, ampicillin (100 g/ml) and, when appropriate,

oNLINe MetHodS

chloramphenicol (30 g/ml) in a 2 ml 96-well block. The corresponding UAAs were also added for experiments requiring the presence of UAAs. These cultures were then grown at 37 C at 1,000 r.p.m. in a benchtop shaker (Vortemp) for 18 h. Cells were then spun down and washed twice with PBS (500 l) and resuspended in PBS (2 ml). All characterization of OFF switch behavior was performed on cells prepared in this manner. For UAA-dependence experiments, samples were grown side by side in the presence or absence of the relevant UAA. For the para-acetylphenylalanine (AcF) concentration dependence experiment, samples were grown in the same 96-well block with various concentrations of AcF. In all cases, cells containing pCCL-000 were also grown in the same 96-well block for the determination of background autofluorescence. UAAs were obtained from the following sources: para-acetylphenylalanine, SynChem; para-azidophenylalanine, Chem-Impex; 4-boronophenylalanine, Sigma-Aldrich; para-iodophenylalanine, Sigma-Aldrich. To characterize ON switch and OR gate behavior, we transformed Top10 cells (Invitrogen) with plasmid pCCL-000 or plasmid pCCL-015; we cotransformed Top10 cells with pCCL-016along with pEVOL-Tyr, pEVOL-pAcF, pEVOL-pAzF, pEVOL-BoF, pEVOL-IF or pEVOL-Dual-pAcF/pAzF. The cells were grown and prepared using the same procedures as those described for the characterization of OFF switch behavior with the following exceptions: during inoculation into 500 l 2YT in 96-well blocks, no l-arabinose was added except where noted; during inoculation into 500 l 2YT in 96-well blocks, 1-methyl-l-tryptophan (100 g/ml, Sigma-Aldrich) was added. For the AcF concentration-dependence experiment, 0.2% l-arabinose was added to induce overexpression of the AcF-specific aaRS so that its saturation is ensured, thus giving a concentration-dependence curve that is based on AcF concentration alone (and not on the aaRS concentration). To prepare cells for the initial screen of tna regulatory region behavior in pCCL-008 through pCCL-018, we used the same procedures as those described for the ON switch behavior except that no washing with PBS was done. Instead, cells in the 2YT growth media were diluted fourfold and used directly for measurements. We note that the OFF switch system corresponding to the pCCL-006 plasmid was tested in E. coli BL21(DE3) cells, and the ON switch system corresponding to plasmids pCCL-015 and pCCL-016 was tested in E. coli Top10 cells. This is because although BL21(DE3) is the optimal strain for using pEVOL plasmids16, when we cotransformed BL21(DE3) cells with pCCL015 or pCCL-016 along with pEVOL plasmids and plated them on selective LB agar plates, no colonies appeared. Due to this BL21(DE3)-specific issue, we carried out experiments involving plasmids pCCL-015 and pCCL-016 in Top10 cells instead. Though we do not understand the reason for this BL21(DE3)-specific incompatibility, we note that there are several differences in the sequence of the chromosomal tna regulatory region of E. coli BL21(DE3) as compared to the sequence of the analogous regulatory region of E. coli K-12 strains (such as Top10). The K-12 strains were the source of the tna regulatory region that has been characterized in the literature and that we used for this study. We also note that the pEVOL plasmids have both constitutive and inducible (induced by l-arabinose) aaRS activities. For the ON switch system, we did not add l-arabinose (except where noted) because we found that constitutive aaRS expression was enough to achieve UAA-dependent ON switch activity. For the OFF switch system, constitutive aaRS expression was not enough to achieve full UAA-dependent OFF switch activity so l-arabinose was added. In addition, we note that when pEVOL plasmids are used for overexpression of proteins containing UAAs, one often notices a lower yield compared to overexpression of the corresponding protein containing no UAAs. This is due to efficiency differences between engineered and natural synthetases as well as the low intracellular availability of certain UAAs11,16. However, in our OFF and ON switches, the UAA-induced activities match the activities resulting from suppression by the natural amino acid tyrosine (Fig. 2). We believe this is because the leader-peptide coding regions in our OFF and ON switches reside in a low-copy vector, thus resulting in a lower demand for the machinery necessary to incorporate UAAs. Finally, we note that in the ON switch experiments, we added 1-methyll-tryptophan, a metabolically stable analog of tryptophan. This was to ensure that when the tna regulatory regions leader peptide was fully translated, the ribosomes tryptophan-binding site would be occupied (if not

2011 Nature America, Inc. All rights reserved.

doi:10.1038/nbt.1741

nature biotechnology

by tryptophan then by 1-methyl-l-tryptophan), thus facilitating stalling over the rut site. Although the addition of 1-methyl-l-tryptophan was not necessary in 2YT because there was excess tryptophan present, we added it nonetheless. Measuring relative GFP expression. Fluorescence of cells containing the superfolder GFP reporter gene under the control of our transcriptional switches was used to determine their activities. First, 200 l of cells prepared according to the cell growth procedures described above were transferred to 96-well plates (Costar 3603). Fluorescence (excitation at 485 nm, emission at 510 nm) and optical densities (600 nm) were then measured using a fluorescence plate reader (Tecan Safire2). The ratio of fluorescence to optical density (RFU/OD) was calculated and the background RFU/OD

corresponding to cells containing pCCL-000, a vector without superfolder GFP, was subtracted where noted. Flow cytometry measurements. Samples prepared according to the cell growth procedures were diluted 250-fold in PBS and analyzed using a flow cytometer (Partec Cyflow Space) in the four parameters of time, forward scatter (FSC), side scatter (SSC) and GFP fluorescence (488 nm excitation, 520 nm band pass emission filter). Data for at least 50,000 cellular counts (triggered by SSC) were collected for each sample. Counts were gated by side and forward scatter. Fluorescence gain was adjusted such that the fluorescence intensity (reported in relative fluorescence units, RFUs) of bacteria containing pCCL000, a plasmid without a GFP gene, centered at ~18 RFUs. Data were processed using FCS Express Version 3.0 (De novo Software).

2011 Nature America, Inc. All rights reserved.

nature biotechnology

doi:10.1038/nbt.1741