post-translational tools expand the scope of synthetic biology

COCHBI-975; NO. OF PAGES 7

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 ([email protected],

[email protected])

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].

Please cite this article in press as: Olson EJ, Tabor JJ. Post-translational tools expand the scope o

www.sciencedirect.com

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



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http://www.sciencedirect.com/science/journal/13675931

2 Synthetic Biology


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



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




Post-translational tools expand the scope of synthetic biology Olson and Tabor 3


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


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



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��].




4 Synthetic Biology


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

+


(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



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






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


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



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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post-translational tools expand the scope of synthetic biology

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