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Post-translational tools expand the scope of synthetic biologyEvan J Olson1 and Jeffrey J Tabor2
Available online at www.sciencedirect.com
Synthetic biology is improving our understanding of and ability
to control living organisms. To date, most progress has been
made by engineering gene expression. However,
computational and genetically encoded tools that allow protein
activity and protein–protein interactions to be controlled on
their natural time and length scales are emerging. These
technologies provide a basis for the construction of post-
translational circuits, which are capable of fast, robust and
highly spatially resolved signal processing. When combined
with their transcriptional and translational counterparts,
synthetic post-translational circuits will allow better analysis
and control of otherwise intractable biological processes such
as cellular differentiation and the growth of tissues.
Addresses1 Graduate Program in Applied Physics, Rice University, 6100 Main
Street, Houston, TX 77005, United States2 Department of Bioengineering, Department of Biochemistry and Cell
Biology, Rice University, 6100 Main Street, Houston, TX 77005,
United States
Corresponding author: Tabor, Jeffrey J (jeff.tabor@gmail.com,
jeff.tabor@rice.edu)
Current Opinion in Chemical Biology 2012, 16:1–7
This review comes from a themed issue on
Synthetic Biology
Edited by Jason W Chin and Lingchong You
1367-5931/$ – see front matter
# 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.cbpa.2012.06.003
IntroductionSynthetic biologists control living cells by dictating when,
where and to what extent gene products, typically
proteins, are active. The simplest strategy for controlling
the activity of a protein is to regulate its concentration,
which is commonly done by varying the rate of transcrip-
tion or translation. In nature, however, post-translational
regulatory processes coordinate biochemical events over
smaller length and time scales, allowing more sophisti-
cated and reliable behaviors to be encoded (Figure 1). For
example, allosteric processes can change protein activity
thousands of fold in milliseconds, while protein–protein
interactions (PPIs) and enzyme cascades can give rise to
switch-like phenotypic changes. Various modes of post-
translational regulation can also buffer the impact of gene
expression noise, making cellular processes more predict-
able [1–9].
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Genetically encoded tools for post-translational control
have lagged behind those for gene expression for several
reasons. First, protein function is, in general, more diffi-
cult to engineer at the level of primary sequence than that
of a promoter or regulatory RNA. Second, gene expres-
sion dynamics occur over experimentally tractable time-
scales of minutes to hours while post-translational
signaling occurs in seconds or less (Figure 2). Finally,
gene expression can often be treated as homogeneous
across an entire cell, while many post-translational pro-
cesses require spatial heterogeneity on the micron scale.
Despite these challenges, recent technological advances
have allowed direct control of protein function in live
cells. In particular, PPIs are now being designed compu-
tationally [10��,11��], and light-switchable proteins
[12,13�] are being used to control an increasing number
of post-translational events in real time. Here, we discuss
new developments and their dual scientific and engin-
eering impact using the example of programmable stem
cell differentiation.
Engineered allosteric control of proteinsPost-translational circuits can be constructed using
proteins that are allosterically regulated by unnatural
inputs [14,15]. In classic work, the autoinhibitory domains
of the cytoskeletal regulator N-WASP were functionally
replaced (modularly recombined) with PDZ and SH3
peptide-binding domains and their ligands [16]. By arran-
ging different numbers of synthetic regulatory modules in
various configurations across the protein, N-WASP was
controlled logically [16] and cooperatively [17] by unna-
tural peptide inputs in vitro. Modular recombination has
since been used to rewire the input [18–21] and output
[22] specificities of kinases, G-protein-coupled receptors
[23], and to construct a synthetic cytoskeletal regulatory
cascade [14].
Allostery can also be engineered by adding a regulatory
domain N-terminal or C-terminal to a protein of interest.
Since its structure was solved in 2003 [24], the blue-light-
switchable light oxygen voltage (LOV) domain from A.sativa Phototropin 1 (AsLOV2) has been used to put many
proteins under optical control [25]. Light absorption by a
flavin mononucleotide (FMN) chromophore triggers dis-
sociation of a C-terminal helix that can de-repress a
sterically or conformationally inactivated output domain.
The ‘lit’ state of AsLOV2 is stable for �30 s, resulting in
reversible switching. In 2008, Strickland et al. demon-
strated that the DNA binding affinity of E. coli TrpR
could be modulated by the fusion of an N-terminal
AsLOV2 domain [26]. This led the way for many other
f synthetic biology, Curr Opin Chem Biol (2012), http://dx.doi.org/10.1016/j.cbpa.2012.06.003
Current Opinion in Chemical Biology 2012, 16:1–7
2 Synthetic Biology
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Figure 1
Perturbation
Temporal SpatialCellular Process
Observation
Concentration
Localization
Interactions
Morphology
Post-TranslationalControl Points
Genetic Control Points
Degradation
Production
Translation
Transcription
(b)
(a)
kmRNA
Modification
Localization
InteractionkI
kP kL
kM
kD
P
t
Current Opinion in Chemical Biology
(a) Perturbative tools allow interrogation and control of cellular processes in both space and time. Gene regulatory responses can be measured as
changes in fluorescent reporter concentration, while post-translational responses can be observed via fluorescent protein localization, FRET, protein
complementation assays (PCAs) or morphological changes. (b) Gene regulation allows temporal control over transcription rate (kmRNA) and translation
rate (kP). Post-translational regulation allows spatial or temporal control of protein degradation (kD), post-translational and conformational modifications
(kM), protein localization (kL), and protein–protein interactions (kI).
LOV-regulated proteins including the histidine kinase
FixL [20], B. subtilis lipase A [27] apoptosis-inducing
Caspase-7 [28], and Rac1 [29��].
Rac1 is a GTPase that binds various effector proteins to
trigger cytoskeletal rearrangements. Hahn and coworkers
fused AsLOV2 to the N-terminus, resulting in a photo-
activated Rac1 (PA-Rac1) with 10-fold increased affinity
for the effector PAK in the ‘lit’ state [29��]. Illumination
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Current Opinion in Chemical Biology 2012, 16:1–7
of 20 mm spots revealed that gradients of Rac1 activity
can polarize mammalian cells, allowing directional move-
ment. Moreover, a spot of Rac1 activation triggers a wave
of RhoA inhibition (observable by FRET) that travels
across the cytoplasm in under 2 min [29��].
Evolution frequently inserts domains in unstructured
protein loops, resulting in novel allosteric control and
new or rewired signaling pathways [30]. Domain insertion
f synthetic biology, Curr Opin Chem Biol (2012), http://dx.doi.org/10.1016/j.cbpa.2012.06.003
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Post-translational tools expand the scope of synthetic biology Olson and Tabor 3
COCHBI-975; NO. OF PAGES 7
Figure 2
Chemical / LightInducible Promoters
(Limited by gene expression)
Transcriptional Regulation
Post-translational Regulation
EngineeredProtein-Protein
Interactions(Limited by diffusion)
EngineeredAllosteric Switching
(Limited by protein switching)
Mammalian
Division TimeGene ExpressionDiffusionProtein Switching
Yeast
E. coli
105 10 4 10 3 10 2 10 1 10 0 10-1
Time (s)
10-2 10-3 10-4 10-5 10-6
Current Opinion in Chemical Biology
Biological processes occur over a wide range of time scales (adapted from [79]). Control of a biological process requires a tool that exerts influence
faster than the process occurs. Devices that control gene expression rate can manipulate processes that occur on the time-scale of minutes. Post-
translational regulatory devices allow access to much faster processes.
is also an effective strategy for engineered allostery [31–34]. In 2003, Ranganathan and coworkers identified ‘sec-
tors’ as coevolving networks of physically interacting
amino acids that ‘wire’ together distal sites of proteins
[35]. Recently, the group inserted AsLOV2 into every
surface site on E. coli dihydrofolate reductase (DHFR)
[36��]. Insertions at 14 of the 70 total sites generate a small
degree of allosteric control, and intriguingly all 14 allo-
steric sites contact the DHFR sector. Although LOV is a
more effective switch when fused to the N-terminus or C-
terminus of a target domain [37], it appears that sectors
could provide the basic road map for engineering allo-
steric control via domain insertion.
Engineered control of protein–proteininteractionsPPIs underlie many cellular regulatory processes. Con-
trolling intracellular and intercellular PPIs is therefore an
important goal for synthetic biology. Along these lines,
sets of orthogonal coiled coil domains have been engin-
eered to heterodimerize with different affinities [38,39].
They have been used to rewire a MAP kinase cascade
[40��], construct transcriptional logic gates [41], and
engineer cooperation between kinesin motor proteins
in vitro [42]. The peptide-binding SH3, GBD, and
PDZ domains [43] and engineered self-assembling
RNA structures [44] have also been used to scaffold
enzymes for improved metabolic flux in bacteria.
Recently, Baker and colleagues designed a novel PPI
from the ‘inside out’ [11��]. An interacting tyrosine/
tryptophan pair was first encoded on the face of the
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Ankyrin Repeat (AR) and P. horikoshii coenzyme A bind-
ing proteins. The pair was then surrounded by a compu-
tationally designed hydrophobic core followed by a
hydrophilic shell. The best interacting pair has a Kd of
130 nM which was improved to a remarkable 180 pM by
directed evolution. Computational design and directed
evolution could be combined to create a sizeable toolbox
of orthogonal PPI parts that could then be linked to
regulatory and catalytic domains to construct synthetic
post-translational circuits. Furthermore, PPI parts with
different affinities could be used to strengthen or weaken
a particular interaction in the network, or even to analyze
natural networks by systematically perturbing PPI
strengths [45].
Switchable protein–protein interactionsSince it was first demonstrated in 1993 [46], the dominant
approach for conditional control of intracellular PPIs has
been the rapamycin-dependent heterodimerization be-
tween FKBP12 and the FRB domain of mTOR. Rapa-
mycin has been used for inducible control of many post-
translational processes [47–56], and photocaged for opti-
cal regulation of kinase signaling [57]. Nonetheless the
molecule diffuses and is not rapidly reversible. To over-
come these limitations, several light-switchable PPI sys-
tems have recently been engineered. These systems use
blue-light induced dimerization between the A. thalianacryptochrome Cry2 and its partner CIB1 [58], LOV-
switchable heterodimerization domains [59], and the
red/far red switchable interaction between A. thalianaphytochrome B (PhyB) and phytochrome interacting
factor 6 (Pif6) [60��].
f synthetic biology, Curr Opin Chem Biol (2012), http://dx.doi.org/10.1016/j.cbpa.2012.06.003
Current Opinion in Chemical Biology 2012, 16:1–7
4 Synthetic Biology
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Figure 3
Phy(a)
(b)
PIF Phy-PIF
650 nm
750 nm
A B
Error signal computation +
-
A-B Interaction
Desired reporterprofile
Observed patterningof reporter
Spatiotemporal patterningof protein state
Projector Camera
+
Current Opinion in Chemical Biology
(a) Phy/PIF system for switchable PPIs, (b) simultaneous optical perturbation and observation of proteins allows feedback-based regulation of
transcription [80] and post-translational processes. The post-translational feedback loop comprises a camera which provides a snapshot of the current
state of a cell which is then compared to the desired state. An error signal generated by this comparison is fed into a light source which projects a
quantitative pattern onto the cells, driving them to the desired state.
The PhyB/Pif6 system (Figure 3a) can control intracellu-
lar protein signaling with seconds-scale reversibility and
subcellular precision [60��]. This feature was recently
exploited to put protein localization and phosphoinositide
signaling under feedback control [61��]. Here, a fluor-
escent reporter of the PPI itself or of phosphoinositide
concentration is measured at the membrane via a micro-
scope-mounted camera and controlled with a light pro-
jector (Figure 3b). Optical feedback can reduce PPI
variations between cells and produce PPI patterns that
vary in time [61��]. This advance is particularly exciting
because it could be used to reverse engineer dynamic
circuit behaviors [62] or precisely control the flow of
signals through engineered circuits even in the face of
gene expression noise and other sources of variability
[61��].
Engineering orthogonal protein circuitsRecently, Kortemme and coworkers engineered an
orthogonal cytoskeletal regulatory pathway by computa-
tionally re-designing the interface between the GTPase
CDC42 and its activator Intersectin (ITSN) [10��]. Repla-
cement of only one ITSN and one CDC42 residue
produced a functional pair with a 480 nM Kd (30 nM for
wild-type) and no cross-talk with wild-type. This
approach can generate new pathways while avoiding
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Current Opinion in Chemical Biology 2012, 16:1–7
unwanted regulatory interactions that may be ported over
using modular recombination. If orthogonal signaling
pairs can be generated from natural versions via small
numbers of mutations in general, existing pathways could
be duplicated and repurposed [15], allowing synthetic
biologists to create many variations on a small number of
themes.
Combining transcriptional and post-translational controlTranscriptional and post-translational devices are being
combined to construct synthetic circuits with expanded
functionality and improved performance [15,40��,63–66,67�]. One application of combined transcriptional/
post-translational circuits is to engineer ultrasensitive
responses, characterized by an effective Hill coefficient
(nH) greater than one [68]. Ultrasensitivity is important in
many switch-like behaviors such as the cell cycle and
differentiation [69], and is also desirable for synthetic
biology [70]. Promoter nH values typically do not exceed 4
or 5, but can be increased by incorporating post-transla-
tional regulation [68]. For example, when a transcrip-
tional activator is stoichiometrically bound by an
inhibitor, a small increase in its concentration can trigger
a large increase in transcription rate [71,72]. Recently, the
mammalian basic leucine zipper (bZIP) transcriptional
f synthetic biology, Curr Opin Chem Biol (2012), http://dx.doi.org/10.1016/j.cbpa.2012.06.003
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Post-translational tools expand the scope of synthetic biology Olson and Tabor 5
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Figure 4
Natural environmentNatural cell
Natural tissueDegree of controlover phenotype
Engineered tissue
Engineered environmentNatural cell
Engineered environmentRewired cell
External control
Engineered environmentEngineered cell
External + internal control
Current Opinion in Chemical Biology
Synthetic biology can contribute to stem cell biology and tissue engineering.
activator CEBPa was expressed with its promoter in S.cerevisiae [67�]. Coexpression of the inhibitor 3HF
increases the activation threshold and nH of CEBPa
activation from 1 to 12, in a manner proportional to its
concentration [72]. Activation threshold, ultrasensitivity,
and magnitude of response can be controlled indepen-
dently by varying the concentrations of kinases and
inhibitors in a MAPK cascade as well [73�].
Applications in cellular differentiationIn the formation of natural tissues, cells integrate chemi-
cal, mechanical, and cell–cell contact cues to regulate
growth, senescence, death, locomotion, and differen-
tiation [74]. Much of this signal-processing and infor-
mation storage is carried out by post-translational
circuits by way of chemical and structural protein modi-
fications [75,76]. Current efforts in stem cell biology and
tissue engineering aim to control signal transduction net-
works with hydrogel microenvironments that mimic those
in vivo [77,78�]. Synthetic biology can contribute to stem
cell biology and tissue engineering on two fronts. First,
natural signaling circuits can be rewired to respond to
tractable inputs such as light as well as, or instead of,
native inputs (Figure 4). Optical signals can then be sent
into the circuits and the flow of biochemical information
can be monitored with FRET/BRET or split-protein
reporters. Then, with an improved understanding of
circuit behaviors, light, and microenvironments can be
used in combination to pattern tissue growth from the top
down. Finally, cellular circuits can eventually be modified
or replaced by streamlined synthetic versions constructed
by engineering, rather than evolutionary processes
(Figure 4). These engineered circuits can encode artificial
developmental programs which, when activated, will
drive cells to grow into a desired tissue with minimal
external intervention. In general, the construction of
integrated transcriptional and post-translational networks
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will allow synthetic biologists to engineer synthetic cel-
lular behaviors that begin to approach the complexity of
those seen in nature.
AcknowledgementsResearch in the Tabor laboratory is supported by the National ScienceFoundation (EFRI-1137266), the Office of Naval Research and the HamillFoundation.
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Current Opinion in Chemical Biology 2012, 16:1–7