Biotechnology Advances 31 (2013) 68–78

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Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Synthetic biology for mammalian cell technology and materials sciences

Raphael J. Gübeli a, b, Katharina Burger a, Wilfried Weber a, b, c, d,⁎
a
Faculty of Biology, University of Freiburg, Schänzlestrasse 1, D-79104 Freiburg, Germany
b
Spemann Graduate School of Biology and Medicine SGBM, University of Freiburg, Albertstrasse 19A, D-79104 Freiburg, Germany
c
Centre for Biological Signalling Studies (BIOSS), University of Freiburg, Hebelstrasse 25, D-79104 Freiburg, Germany
d
FIT Freiburg Center for Interactive Materials and Bio-Inspired Technologies, University of Freiburg, Germany

a r t i c l e i n f o a b s t r a c t

Available online 21 January 2012 The synthetic reconstruction of natural gene networks and the de novo design of artificial genetic circuits
provide new insights into the cell's regulatory mechanisms and will open new opportunities for drug discovery
Keywords: and intelligent therapeutic schemes. We will present how modular synthetic biology tools like repressors, pro-
Biohybrid material moters and enzymes can be assembled into complex systems in order to discover small molecules to shut
Drug depot
off antibiotic resistance in tubercle bacteria and to design self-sufficient therapeutic networks. The transfer
Hydrogel
of these synthetic biological modules to the materials science field enables the construction of novel drug-
Stimulus-responsive
Synthetic gene network
inducible biohybrid materials for biomedical applications.
© 2012 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
2. The diversity of synthetic biological modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
2.1. Stimulus-responsive protein-DNA interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
2.2. Stimulus-responsive protein dimerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3. Towards therapeutic applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.1. Drug discovery platform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.2. Synthetic therapeutic prostheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
4. Synthetic biology for the synthesis of interactive biohybrid materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
4.1. Antibiotic-responsive biohybrid materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4.2. Drug-responsive humanized biohybrid materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4.3. Drug-responsive hydrogels based on DNA-protein interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
5. Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

1. Introduction robust functionalities. Initially, such designed systems were tested in

Synthetic biology aims to advance life sciences by the application
of engineering approaches in order to construct novel biological sys-
tems exerting complex functions. Following a bottom-up strategy,
single well-characterized modules are assembled into complex biological
systems guided by mathematical modeling leading to predictable and
⁎ Corresponding author at: Schänzlestrasse 1, D-79104 Freiburg. Tel.: + 49 761 203
97654; fax: + 49 761 203 97660.
E-mail address: wilfried.weber@biologie.uni-freiburg.de (W. Weber).
prokaryotes (Basu et al., 2005; Elowitz and Leibler, 2000) mainly due
to simple genetic background and manipulation. As a next step, the
focus was laid on establishing these circuits in higher-order organisms
from yeast (Ajo-Franklin et al., 2007) to plants (Antunes et al., 2006)
up to mammalian cells as they better represent the complexity in
humans.
We will describe how simple, well-characterized biological modules
can be combined into higher-order synthetic networks; in vivo, for the
development of therapeutic genetic circuits and, in vitro for the syn-
thesis of interactive biohybrid materials for biomedical applications.

0734-9750/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2012.01.007
 R.J. Gübeli et al. / Biotechnology Advances 31 (2013) 68–78 69

2. The diversity of synthetic biological modules
All higher-order networks in synthetic biology rely on the combi-
nation of well-characterized modules out of a library of molecular
switches being initially designed mainly for controllable transgene
expression. These switches mostly depend on molecular binding
events such as protein-DNA or protein–protein interactions which
are rendered stimulus-responsive either by the allosteric binding of
small molecules such as drugs, metabolites or hormones or by exter-
nal physical inputs such as temperature and light (Weber et al.,
2003; Yazawa et al., 2009 and extensively reviewed by Weber and
Fussenegger, 2011). A large variety of such conditional protein–DNA
and protein–protein interactions has been characterized recently which
allows for the combination of these switches into complex biological
circuits.
2.1. Stimulus-responsive protein-DNA interactions
The conditional, site-specific interaction of proteins with their DNA
recognition sequence represents one approach to confer stimulus-
responsiveness to molecular switches (Fig. 1). Commonly, the DNA-
binding protein belongs to the large family of DNA repressor proteins
which are allosterically controlled by a single effector substance in a
very selective and quantitative manner, as they evolved e.g. to control
the metabolic state or to respond to potentially harmful environmental
conditions in bacteria (Ramos et al., 2005).
The construction of controllable, synthetic transgene expression
systems by the fusion of such trigger-inducible DNA-binding proteins
with transcriptional activator (Fig. 1a) or silencer (Fig. 1b) domains
allowed for a controllable on- and off-situation of promoters functio-
nalized with single or multiple repeats of the corresponding operator
sequences. This concept was pioneered by Gossen and Bujard (1992)
building an inducible mammalian gene expression system based on
the exogenous Tet repressor (TetR) fused to the activation domain
of the virion protein 16 of the Herpes simplex virus (VP16) leading
to a tetracycline-responsive hybrid transactivator. In order to allow
for the simultaneous control of several target genes, this switch con-
cept has been extended using repressor proteins responsive to other
classes of antibiotics such as streptogramins (Fussenegger et al., 2000)
or macrolides (Weber et al., 2002) allowing for the simultaneous con-
trol of several target genes.
2.2. Stimulus-responsive protein dimerization
Protein binding partners whose interaction can be influenced by
the addition of a dimerizing or monomerizing substrate represent
the most versatile way to confer stimulus responsiveness to a syn-
thetic system. For in vivo applications, most stimulating compounds
are of exogenous origin allowing for a precise regulation without eli-
citing systemic perturbations.
Such a widely used controllable protein switch was based on the
gyrase B (GyrB) protein from Escherichia coli being responsive to
different forms of coumarin antibiotics (Ali et al., 1993, Fig. 2a). The
addition of coumermycin led to homodimerization of GyrB whereas
subsequently applied novobiocin competitively replaced coumermycin
from the GyrB binding pocket leading to the monomerization of the
protein. However, as GyrB is of bacterial origin and might therefore
elicit harmful human immune responses, a system based on the
rapamycin-mediated heterodimerization of the human-derived FK506-
binding protein (FKBP) and the 11 kDa-domain of the FRAP-protein
(FRB), was developed (Rivera et al., 1996, Fig. 2b). A single point
mutation in the amino acid sequence converted the normally monomeric

Fig. 1. Molecular switches based on protein-DNA interactions. (a) Tetracycline-repressible expression system. The tetracycline repressor TetR fused to the transactivation
domain of the virion protein 16 of the Herpes simplex virus (VP16) binds to a 7-fold repeat of its cognate operator sequence (tetO7) upstream of a minimal promoter (Pmin).
The RNA polymerase is recruited and activates the expression of the gene of interest (goi). The addition of tetracycline that binds to the TetR fusion protein leads to the disso-
ciation from tetO7, prevents RNA polymerase recruitment and therefore abolishes goi expression. (b) Tetracycline-inducible expression. TetR binds to a 2-fold operator sequence
(tetO2) downstream of a constitutive promoter of the human cytomegalovirus thereby inhibiting RNA polymerase progression. Addition of tetracycline leads to the dissociation
of TetR from tetO2 thereby enabling RNA polymerase read-through and goi expression. pA, polyadenylation signal.
70 R.J. Gübeli et al. / Biotechnology Advances 31 (2013) 68–78

Fig. 2. Molecular switches based on protein–protein interactions. (a) Coumarin antibiotic-switchable protein dimerization. By the addition of coumermycin, the N-terminal domain
of the gyrase B (GyrB) from E. coli is dimerized. An excess of novobiocin competitively replaces coumermycin from the GyrB binding pocket thereby leading to GyrB monomer for-
mation. (b) Rapamycin-switchable protein dimerization. The human FK506-binding protein (FKBP) is heterodimerized with the 11 kDa-domain of the FRAP-protein (FRB) by the
addition of rapamycin. Withdrawal of rapamycin returns the proteins to monomeric state. (c) FK506-switchable protein dimerization. A variant of FKBP harboring a single point
mutation (FM) spontaneously forms stable dimers. The ligand FK506 binds to the FM protein thereby resulting in protein monomerization.

FKBP to the dimeric FM variant which could be converted back to the mo-
nomeric state by the ligand FK506 (Rollins et al., 2000, Fig. 2c).
These protein–protein interactions have been applied in vivo to
control the activity of enzymes and promoters, signaling cascades
and protein secretion. For example, enzyme activity was controlled
by fusing three GyrB domains to the chlamydial protease-like activity
factor (CPAF) in order to restore the human disease phenotype of the
intracellular pathogen Chlamydia trachomatis in human cells (Paschen
et al., 2008, Fig. 3a). By the addition of coumermycin leading to GyrB
dimerization and thereby bringing CPAF moieties close together,
also referred as induced proximity, they showed that CPAF is able
to induce its proteolytic activity by autocatalytic cleavage leading
to digestion of important cell cycle factors such as cyclin B1 and fi-
nally nonapoptotic cell death. The synthetic, inducible GyrB-CPAF
fusion construct proved that CPAF is the main disease factor of a
C. trachomatis infection.
Inducible gene expression was achieved by fusing three FKBP do-
mains to a synthetic zinc-finger DNA-binding domain (ZFHD1) binding
to a minimal promoter carrying 12 ZFHD1 operator elements (Rivera et
al., 1996, Fig. 3b). Induction of target gene expression was achieved by
the rapamycin-mediated binding to an FRB fusion protein containing
the p65 transcriptional activator domain of the human transcription
factor NF-κB. The change of single components of this system, e.g. the
development of synthetic, more bioavailable dimerizers (Amara et al.,
1997) or the conversion of normally monomeric FKBP to the dimeric
FM protein (Rollins et al., 2000), showed that one can modify these sys-
tems in a very versatile manner.
Signaling events were artificially controlled by triggering recep-
tor oligomerization intracellularly (Spencer et al., 1993, Fig. 3c).
The fusion of three FKBP motifs to a ζ chain of the T cell antigen
receptor (TCR) harboring tyrosine activation motifs together with
a myristylation motif of c-Src (Src) anchored the chimeric receptor
in the cell membrane and made it responsive to the cell-permeable syn-
thetic ligand FK1012 being a FK506 dimer. Addition of FK1012 brought
the ζ chains in close proximity which resulted in a TCR-like signaling
cascade mediated by tyrosine kinases.
Secretion was engineered to allow for fast, burst-like cellular protein
secretion resembling the behavior of insulin released by secretory ves-
icles of β-cells in vivo (Rivera et al., 2000, Fig. 3d). The expression of
human proinsulin linked to several repeats of the FM protein led to
intracellular aggregates of proinsulin-FM in the endoplasmic reticu-
lum (ER). Administration of the cell-permeable inducer FK506 resulted
in immediate dissolution of the aggregates, transport to the Golgi
compartment, further cleavage of the linker by a Golgi-specific furin
protease and finally secretion of native insulin.
3. Towards therapeutic applications
The above-mentioned synthetic biological switches were assembled
into genetic networks suitable for biomedical applications. Here we
review artificial genetic networks that can be used for the discovery
of anti-infective drugs (Weber et al., 2008), that act as molecular
prostheses in order to improve cow fertilization by an open-loop
gene network (Kemmer et al., 2011) or to detect a disease state and
autonomously trigger a therapeutic response (Kemmer et al., 2010),
which might be of high value to treat complex genetic or acquired
disorders.
 R.J. Gübeli et al. / Biotechnology Advances 31 (2013) 68–78 71

3.1. Drug discovery platform
A synthetic mammalian gene network was applied for the iden-
tification of small molecules that overcome the intrinsic resistance
of Mycobacterium tuberculosis to the antibiotic ethionamide. In tubercle
bacteria, ethionamide is activated by the Baeyer-Villiger monooxy-
genase EthA to a cytotoxic compound. However, this activation is
rather inefficient as the gene encoding EthA is repressed by the
TetR-like repressor EthR. In order to increase EthA production and
the conversion of ethionamide to a cytotoxic compound, a mammalian

Fig. 3. Applications of protein–protein switches. (a) Protease activation by inducible proximization. The chlamydial protease-like activity factor (CPAF) is fused to a triple repeat
of GyrB. Addition of coumermycin leads to autocatalytic cleavage, dimerization and induction of the protease activity of CPAF resulting in the degradation of target proteins.
(b) Rapamycin-inducible gene expression. Three FKBP domains are fused C-terminally to the synthetic DNA-binding domain ZFHD1BD which binds to a 12-fold repeat of the
operator sequence (ZFHD)12 located upstream of a minimal cytomegalovirus-derived promoter (Pmin). A chimeric factor consisting of FRB fused to the p65 transcriptional
activator domain of the human transcription factor NF-κB is coexpressed. Addition of rapamycin leads to heterodimerization of the FKBP and FRB subunits thereby recruiting
the RNA polymerase by the p65 domain and allowing goi expression. (c) Inducible intracellular signal transduction. A chimeric intracellular receptor is constructed by fusing
the ζ chain of the T cell antigen receptor harboring tyrosine activation motifs with a myristylation motif of c-Src (Src) for membrane targeting and a triple repeat of FKBP.
Addition of the cell-permeable synthetic FK506 dimer (FK1012) leads to dimerization of FKBP and brings the ζ chain in proximity thereby mimicking antigen-induced TCR
oligomerization which results in tyrosine kinase-mediated downstream signaling. (d) Inducible protein secretion. When human proinsulin (insulin) is fused via a furin cleavage
site (blue) to a 4-fold repeat of the FM protein and expressed in mammalian cells, the FM domains spontaneously dimerize leading to aggregation of the whole protein in the
endoplasmic reticulum (ER). Upon administration of the ligand FK506, the FM domains monomerize which results in further transport of the fusion protein to the Golgi com-
partment, processing by furin proteases and further secretion of native insulin.
72 R.J. Gübeli et al. / Biotechnology Advances 31 (2013) 68–78
Fig. 3 (continued).
screening system was developed to identify compounds that inactivate
the repressor EthR. (Weber et al., 2008, Fig. 4a). Therefore, EthR was
fused to the VP16 transactivation domain. This chimeric transactivator
bound a minimal promoter harboring the OethR DNA-binding sequence
thereby inducing the expression of a reporter gene. A screen for possible
effector substances revealed that the non-toxic, cell-permeable food
additive 2-phenylethyl-butyrate was able to abolish the EthR- OethR
interaction thereby potentiating the therapeutic efficacy of ethion-
amide when administered together. In principle, this screening
setup could be extended to other pathogenic resistance mechanisms
relying on DNA-binding proteins in order to reveal other factors for
powerful co-treatment regimes with established drugs.
3.2. Synthetic therapeutic prostheses
An open-loop gene network has been developed for precise tempo-
ral regulation of bovine artificial insemination (Kemmer et al., 2011,
Fig. 4b). Today, the efficacy of artificial cow insemination is still limited
due to the difficulty to find the exact point in time during the cow estrus
for sperm administration. A possible solution would be a system which
allows for synchronization of sperm release with estrus-associated
hormone changes. For this aim, a molecular switch sensitive to the
ovulation-associated luteinizing hormone (LH) has been established
in human embryonic kidney cells: upon increasing LH levels, a heter-
ologously expressed rat luteinizing hormone receptor (LHR) triggered
a G protein-coupled receptor (GPCR) response leading to increased
intracellular cAMP levels acting on a cAMP-responsive promoter to
produce a secreted cellulase derived from Bacillus subtilis. When these
cells transgenic for the LH switch were encapsulated together with
bull sperms into cellulose microcapsules, an LH increase led to the
cellulase-mediated breakdown of the capsules thereby releasing
the sperms at the optimum point in time for the fertilization of oo-
cytes in the cows.
A closed-loop control switch that senses a disease state and autono-
mously triggers a therapeutic response has recently been developed by
Kemmer et al. (2010, Fig. 4c) as a prototype therapy against gouty ar-
thritis. Unlike other mammals, humans lack a functional urate oxi-
dase therefore leading to high urate serum levels. This is beneficial
on the one hand as urate is an efficient radical scavenger, however,
it is also detrimental as urate might precipitate into harmful urate
crystals in the body in gouty arthritis. As a therapeutic option, a sys-
tem which autonomously senses and degrades excess urate concen-
trations while leaving physiological levels unaffected, was constructed
based on the transcriptional repressor HucR. HucR, a urate-sensitive re-
pressor protein from Deinococcus radiodurans, was fused to the transsi-
lencing domain of the Krueppel-associated box (KRAB) protein. Binding
to a constitutive promoter harboring an octameric repeat of the HucR
operator sequence hucO shut off expression of the therapeutic trans-
gene, an Aspergillus flavus-derived secreted urate oxidase (Fig. 4c).
However, at pathologically high urate concentrations, HucR was disso-
ciated from hucO thereby leading to the expression of urate oxidase
that finally degraded excess uric acid until physiological levels were re-
stored. The functionality of this system was demonstrated by microen-
capsulating and implanting cells with this designer circuit into a mouse
model of gouty arthritis. It was shown that this system was able to de-
grade excess uric acid while leaving physiological levels unaffected.
4. Synthetic biology for the synthesis of interactive biohybrid
materials
Although genetic circuits were shown to be reliably controllable
on a molecular level and inherently functional under physiological
 R.J. Gübeli et al. / Biotechnology Advances 31 (2013) 68–78 73

conditions, their human therapeutic use remains associated with safety

concerns due to the transfer of genetic elements into a human being
having the potential of severe side-effects (Hacein-Bey-Abina et al.,
2003). a
One possible way to overcome these concerns is to transfer the
well-characterized molecular tools and regulation modules out of
the cellular context to a cell-free system in the form of a biohybrid
material. In a pioneering study, Ehrbar et al. (2008) transferred the
pharmacologic principle of the GyrB-coumarin antibiotic interaction
(Fig. 2a) to the materials science field by the construction of a biohy-
brid material for the drug-inducible release of incorporated biophar-
maceuticals. Following this first successful crossover of synthetic
biology with material sciences, follow-up studies emerged with fur-
ther humanized hydrogel components (Kämpf et al., 2010) or a gen-
eral approach to synthesize biohybrid materials responsive to a
desired stimulus (Christen et al., 2011) (Fig. 5).

4.1. Antibiotic-responsive biohybrid materials

In search of a biohybrid material responsive to a stimulus that

was biocompatible and worked under physiological conditions, Ehrbar
et al. (2008) applied the GyrB-coumarin switch (Fig. 2a) to develop
a hydrogel responsive to physiologically compatible levels of amino-
coumarin antibiotics (Fig. 5a). For this aim, linear polyacrylamide
which is commonly used in biomedical applications (Caulfield et al.,
2003; Narins et al., 2010; Pallua and Wolter, 2010) was functionalized
with Ni 2 +-chelating nitrilotriacetic acid (NTA) groups and grafted via
a hexahistidine tag to GyrB. Addition of coumermycin triggered GyrB
dimerization thereby crosslinking the polymer resulting in the for-
mation of a hydrogel. Administration of increasing concentrations
of the coumarin antibiotic novobiocin however, led to the competi-
tive replacement of coumermycin thereby resulting in a dose- and
time-adjustable dissolution of the hydrogel. The suitability as a
stimulus-responsive drug depot was shown by the incorporation of
the model therapeutic protein vascular endothelial growth factor
(VEGF) that was released from the hydrogel in a spatiotemporally
regulated manner and proved to be bioactive after release as shown
by its growth-promoting effect on human umbilical vein endothelial
cells (HUVECs).

4.2. Drug-responsive humanized biohybrid materials

In order to avoid possible immunogenic effects of the bacteria-
derived GyrB upon in vivo application, a humanized hydrogel was
developed by Kämpf et al. (2010, Fig. 5b) by functionalizing an acryl-
amide polymer with the human-derived homodimerizing FM protein
(see Fig. 2c). The dose- and time-adjustable dissolution kinetics was
achieved by administration of increasing concentrations of the FM-
monomerizing ligand FK506. When implanted subcutaneously into
mice, these hydrogels were dissolved by the oral or intraperitoneal
administration of the inducer FK506 thereby releasing the incorpo-
rated cargo protein VEGF into the blood serum. This study was the
first in vivo demonstration of a hydrogel releasing biopharmaceuti-
cals in response to a small-molecule inducer.
4.3. Drug-responsive hydrogels based on DNA-protein interactions
While the above-described hydrogels were based on protein-
protein interactions, the conditional interaction of the DNA-binding
protein TetR with its corresponding DNA operator sequence tetO
(Fig. 1) was used by Christen et al. (2011, Fig. 5c) to synthesize a bio-
hybrid material responsive to tetracycline. The hydrogel was based
on differentially functionalized acrylamide polymers where one
was modified with the tetO motif and the other with a single-
chain variant of the TetR repressor. Upon mixing both polymers,
a hydrogel formed immediately. The hydrogel was dissolved in a
Fig. 4. Application of synthetic networks for drug discovery and therapy. (a) Synthetic
gene network for the discovery of anti-tuberculosis drugs. A screening system is designed
in mammalian HEK293-T cells based on the finding that, in Mycobacterium tuberculosis,
the TetR-family protein EthR is involved in the resistance to ethionamide. EthR is fused
to the transactivator domain VP16 and binds to its operator site (OethR) upstream of a min-
imal promoter of the Drosophila heatshock protein 70 (Phsp70min) thereby activating the
expression of the reporter gene secreted alkaline phosphatase (seap). This system enables
the screening for substances that are cell-permeable and additionally bind to the EthR-
VP16 fusion protein leading to dissociation from OethR and thereby decreasing SEAP pro-
duction. Increasing amounts of the compound 2-phenylethyl-butyrate lead to a gradual
decrease in SEAP reporter production in cells harboring the screening gene network
(EthRHEK-SEAP cells) while not showing cytotoxic effects when the compound is tested
on cells constitutively expressing SEAP (HEK-SEAP cells). (b) Open-loop controlled gene
network for artificial cow insemination. Cow sperms are trapped together with effector
HEK293-T cells harboring an ovulation-responsive gene network and implanted into cows.
In the presence of increased levels of ovulation-associated bovine luteinizing hormone
(bLH), the rat luteinizing hormone receptor (rLHR) recombinantly expressed on the effector
cells is stimulated thereby activating a G protein-coupled receptor response. This increases
cAMP concentrations that activate the cAMP-responsive promoter (PCRE4) resulting in the
expression of a secreted cellulase from Bacillus subtilis which finally degrades the capsule
wall and releases the sperms. (c) Closed-loop control gene network for the management
of urate homeostasis. The repressor HucR from Deinococcus radiodurans is fused to the trans-
silencing domain of the Krüppel-associated box (KRAB) protein and expressed in HeLa cells.
By binding to an 8-fold repeat of its operator sequence (hucO8) downstream of the constitu-
tive promoter from Simian virus 40 (PSV40), RNA polymerase progression is stopped and the
secretion-engineered urate oxidase (smUox) is not produced. When the urate concentration
rises from physiological (green) up to pathological (red) levels, HucR is dissociated from
hucO8 thereby leading to smUox expression and secretion which degrades the excess in
urate back to the physiological level. In order to facilitate urate uptake by the cell, the
urate transporter URAT1 is expressed.
74 R.J. Gübeli et al. / Biotechnology Advances 31 (2013) 68–78

Fig. 4 (continued).

dose-adjustable manner by the administration of tetracycline. In order
to be able to incorporate different kinds of biomolecules as cargo, the
protein A domain from Staphylococcus areus was incorporated in the
hydrogel as an anchor protein for specifically binding proteins har-
boring an Fc-region derived from immunoglobulin G. This approach
has been validated by the incorporation and dose-dependent release
of an Fc-tagged version of the human cytokine interleukin 4 (IL-4Fc).
The released cytokine was shown to be fully bioactive in a mammalian
cell-based assay.
This hydrogel concept has the potential to act as a general plat-
form for the synthesis of biohybrid materials tailored to sense a de-
sired stimulus by simply exchanging the TetR/tetO pair with another
protein/DNA combination out of the large family of allosterically regu-
lated DNA-binding proteins (Ramos et al., 2005).
 R.J. Gübeli et al. / Biotechnology Advances 31 (2013) 68–78 75

c physiological [Urate] pathological [Urate]

Urate

Urate
URAT1 URAT1

RNA RNA sm
Pol HucR HucR Pol Uox

PSV40 hucO8 smUox pA PSV40 hucO8 smUox pA

AAAAA

sm sm
Uox Uox

HeLa HeLa

physiological pathological pathological

smUox

[Urate]

physiological

0 12
[Urate] Time [d]
Fig. 4 (continued).

5. Conclusions and outlook A direct human therapeutic application of these systems in a

A large variety of genetic switches has been established in mam-
malian cells in the past two decades. Combination of these switches
enabled the construction of complex, controllable networks that show
a broad application potential as described for the biomedical area. In
the near future, drug screening platforms based on several of these
synthetic networks will allow for the finding of more reliable initial
drug candidates and improved functional characterization thereof,
thereby increasing the success rate of drug discovery and develop-
ment in the pharmaceutical industry (Weber and Fussenegger, 2009).
In basic research, the study of complex dynamic processes and
fundamental concepts can be simplified by reverse engineering and
proof of these phenomena with synthetic gene networks. This has al-
ready been successfully demonstrated by a study where signaling cas-
cades were artificially rewired in order to show B cell antigen
receptor oligomerization (Yang and Reth, 2010).
gene therapy approach still faces unsolved challenges as the inte-
gration of these networks into a genome can drastically affect the
properties of the synthetic network but can also be harmful to the
recipient cell by perturbing the transcriptional control machinery.
Currently, important steps are being made towards the improve-
ment of existing molecular tools for the site-specific integration of
genetic material into a “safe harbor” in the human genome
(Händel and Cathomen, 2011; Naldini, 2011) which will allow for
safe, side effect-free and reproducible action of genome-integrated
therapeutic networks in the future.
A further option to maintain the regulatory potential of the synthetic
genetic switches and networks while circumventing the challenges as-
sociated with therapeutic gene transfer, would be the use of a cell-free
matrix as described for the synthetic biology-derived biohybrid mate-
rials. This would allow combining the advantages of the diverse, exten-
sively characterized biological devices from synthetic biology with
76 R.J. Gübeli et al. / Biotechnology Advances 31 (2013) 68–78

materials science providing a huge variety of polymer chassis already References

proven safe in several therapeutic applications.
Considering the recent development in synthetic biology-inspired
hybrid materials reaching a critical mass of well-characterized modules
that can be combined to build novel complex and dynamic networks,
this field holds great promise to grow from inducible drug delivery
devices and tissue engineering scaffolds towards a platform for the
next generation of intelligent, self-sufficient therapeutic treatments.
Acknowledgments
Work in the group of WW is supported by the European Re-
search Council under the European Community's Seventh Frame-
work Programme (FP7/2007-2013)/ERC Grant agreement no. 259043-
CompBioMat and the Excellence Initiative of the German Federal and
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 R.J. Gübeli et al. / Biotechnology Advances 31 (2013) 68–78 77

GEL SOL
b
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FK506
+
FK506

FM FM
VEGF
FM

FM
FM

VEGF
VEGF

FM

FM
VEGF

FM
in vivo released VEGF

- FK506 + FK506

Fig. 5 (continued).

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78 R.J. Gübeli et al. / Biotechnology Advances 31 (2013) 68–78

c GEL SOL
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+
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protein A

IL-4FC scTetR

Tetracycline : TetR
3:3 HEK293-T
IL-4FC release

2:3 P
1:3 STAT6 RNA
Pol
0:3
Peotaxin3 seap pA

AAAAA

SEAP

0 25
Time [h]
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IL-4 Standard
Released IL-4FC
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[IL-4]
Fig. 5 (continued).

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