letters
Regulation of transcription by unnatural amino acids
© 2011 Nature America, Inc. All rights reserved.
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 logics3–8. 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 operon’s 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 trypto­
phan 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 factor–dependent 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 factor–mediated 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 codes—these 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
expansion11—the number of UAA-induced switches that could ­readily
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. ([email protected]) or A.P.A. ([email protected]).
Received 23 July 2010; accepted 1 December 2010; published online 16 January 2011; doi:10.1038/nbt.1741
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letters
UAAs
Control, sensing
- 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
© 2011 Nature America, Inc. All rights reserved.
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
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 homo­
geneous 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 operon’s 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 operon–sensing 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
nature biotechnology VOLUME 29 NUMBER 2 FEBRUARY 2011
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 peptide’s 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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2,000
1,500
100
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50
500
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p
pE CC
VO L-0
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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 25–70% 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.
166
(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 factor–dependent 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-016’s 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 peptide’s 12 C-terminal
amino acids and not by interactions with the earlier positions in the
peptide where our UAAs are incorporated19. Therefore, the function
a
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© 2011 Nature America, Inc. All rights reserved.
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VOLUME 29 NUMBER 2 FEBRUARY 2011 nature biotechnology
© 2011 Nature America, Inc. All rights reserved.
letters
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 acti­
vities 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 aaRSs—we cloned
such a plasmid, pEVOL-Dual-pAcF/pAzF—we 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)21–25, 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.
nature biotechnology VOLUME 29 NUMBER 2 FEBRUARY 2011
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 β2-adrenergic receptor, RAR-β2 and
AdoMetDC translational regulation in mammals)28–30 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
control—and more generally the development of scalable, homogeneous, composable and connectable components26—will 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/.
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168
VOLUME 29 NUMBER 2 FEBRUARY 2011 nature biotechnology
© 2011 Nature America, Inc. All rights reserved.
ONLINE METHODS
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 µg/ml) and, in the case of cells also containing pEVOL plasmids, chloramphenicol (30 µg/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 µg/ml) and chloramphenicol (30 µg/ml)
when appropriate. These cultures were shaken overnight at 200 r.p.m.
at 37 °C. We used 5 µl of each overnight culture to inoculate 500 µl of 2YT
containing 0.2% l-arabinose, ampicillin (100 µg/ml) and, when appropriate,
doi:10.1038/nbt.1741
­chloramphenicol (30 µg/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 µl) 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 rele­
vant 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 µl 2YT in 96-well blocks, no l-arabinose was added
except where noted; during inoculation into 500 µl 2YT in 96-well blocks,
1-methyl-l-tryptophan (100 µg/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 region’s leader peptide was fully translated, the ribosome’s tryptophan-binding site would be occupied (if not
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.
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.
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 µl 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.
nature biotechnology
doi:10.1038/nbt.1741