NATURE BIOTECHNOLOGY VOLUME 27 NUMBER 12 DECEMBER 2009 1139
Synthetic gene networks: what have we learned and what do
we need?
The engineering of mechanical, electrical and chemical systems is
enabled by well-established frameworks for handling complexity, reli-
able means of probing and manipulating system states and the use of
testing platforms—tools that are largely lacking in the engineering of
biology. Developing properly functioning biological circuits can involve
complicated protocols for DNA construction, rudimentary model-
guided and rational design, and repeated rounds of trial and error fol-
lowed by fine-tuning. Limitations in characterizing kinetic processes
and interactions between synthetic components and other unknown
constituents in vivo make troubleshooting and modeling frustrating
and prohibitively time consuming. As a result, the design cycle for engi-
neering synthetic gene networks remains slow and error prone.
Fortunately, advances are being made in streamlining the physical
construction of artificial biological systems, in the form of resources
and methods for building larger engineered DNA systems from smaller
defined parts
22,30–32
. Additionally, large-scale DNA sequencing and
synthesis technologies are gradually enabling researchers to directly
program whole genes, genetic circuits and even genomes, as well as to
re-encode DNA sequences with optimal codons and minimal restric-
tion sites (see review
33
).
Despite these advances in molecular construction, the task of build-
ing synthetic gene networks that function as desired remains extremely
challenging. Accelerated, large-scale diversification
34
and the use of
characterized component libraries in conjunction with in silico mod-
els for a priori design
22
are proving useful in helping to fine-tune net-
work performance toward desired outputs. Even so, in general, synthetic
biologists are often fundamentally limited by a dearth of interoper-
able and modular biological parts, predictive computational modeling
capabilities, reliable means of characterizing information flow through
engineered gene networks and test platforms for rapidly designing and
constructing synthetic circuits.
In the following subsections, we discuss four important research
efforts that will improve and accelerate the design cycle for next-gener-
ation synthetic gene networks: first, advancing and expanding the tool-
kit of available parts and modules; second, modeling and fine-tuning
Ten years since the introduction of the field’s inaugural devices—the
genetic toggle switch (J.J.C. and colleagues)
1
and repressilator
2
—
synthetic biologists have successfully engineered a wide range of
functionality into artificial gene circuits, creating switches
1,3–9
, oscil-
lators
2,10–12
, digital logic evaluators
13,14
, filters
15–17
, sensors
18–20
and
cell-cell communicators
15,19
. Some of these engineered gene networks
have been applied to perform useful tasks such as population con-
trol
21
, decision making for whole-cell biosensors
19
, genetic timing for
fermentation processes (J.J.C. and colleagues)
22
and image process-
ing
23–25
. Synthetic biologists have even begun to address important
medical and industrial problems with engineered organisms, such
as bacteria that invade cancer cells
26
, bacteriophages with enhanced
abilities to treat infectious diseases (T.K.L. and J.J.C.)
27,28
, and yeast
with synthetic microbial pathways that enable the production of
antimalarial drug precursors
29
. However, in most application-driven
cases, engineered organisms contain only simple gene circuits that
do not fully exploit the potential of synthetic biology. There remains
a fundamental disconnect between low-level genetic circuitry and
the promise of assembling these circuits into more complex gene
networks that exhibit robust, predictable behaviors.
Thus, despite all of its successes, many more challenges remain in
advancing synthetic biology to the realm of higher-order networks
with programmable functionality and real-world applicability. Here,
instead of reviewing the progress that has been made in synthetic
biology, we present challenges and goals for next-generation syn-
thetic gene networks, and describe some of the more compelling
circuits to be developed and application areas to be considered.
Next-generation synthetic gene networks
Timothy K Lu
1–3
, Ahmad S Khalil
3
& James J Collins
3,4
Synthetic biology is focused on the rational construction of biological systems based on engineering principles.
During the field’s first decade of development, significant progress has been made in designing biological parts and
assembling them into genetic circuits to achieve basic functionalities. These circuits have been used to construct
proof-of-principle systems with promising results in industrial and medical applications. However, advances in
synthetic biology have been limited by a lack of interoperable parts, techniques for dynamically probing biological
systems and frameworks for the reliable construction and operation of complex, higher-order networks. As these
challenges are addressed, synthetic biologists will be able to construct useful next-generation synthetic gene
networks with real-world applications in medicine, biotechnology, bioremediation and bioenergy.
1
Department of Electrical Engineering and Computer Science, Massachusetts
Institute of Technology, Cambridge, Massachusetts, USA.
2
Harvard-MIT
Health Sciences and Technology, Cambridge, Massachusetts, USA.
3
Howard
Hughes Medical Institute, Department of Biomedical Engineering, Center
for BioDynamics, and Center for Advanced Biotechnology, Boston University,
Boston, Massachusetts, USA.
4
Wyss Institute for Biologically Inspired
Engineering, Harvard University, Boston, Massachusetts, USA. Correspondence
should be addressed to T.K.L. (timlu@mit.edu).
Published online 9 December 2009; doi:10.1038/nbt1591
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1140 VOLUME 27 NUMBER 12 DECEMBER 2009 NATURE BIOTECHNOLOGY
rationally programmed based on sequence specificity
7,40,41
. Novel
circuit interconnections could be established using small interfering
RNAs (siRNAs) to control the expression of specific components.
Recombinases, which target specific DNA recombinase-recognition
sites, also represent a fruitful, underutilized source of interoperable
parts. Recombinases have been used in the context of synthetic biol-
ogy to create memory elements and genetic counters
9
. However, more
than 100 natural recombinases are known, and these can be engi-
neered by mutagenesis and directed evolution for greater diversity
and sequence specificity
42–45
.
Libraries of well-characterized, interoperable parts, such as tran-
scription factors and recombinases, would vastly enhance the ability
of synthetic biologists to build more complex gene networks with
greater reliability and real-world applicability. In addition to libraries
of individual parts, it would be of great value to have well-characterized
and interoperable modules (e.g., switches, oscillators and interfaces)
that could be used in a plug-and-play fashion to create higher-order
networks and programmable cells. As the number of parts and mod-
ules expands, high-throughput, combinatorial efforts for quantifying
the levels of interference and cross-talk between multiple components
within cells will be increasingly important as guides for choosing the
most appropriate components for network assembly.
Modeling and fine-tuning synthetic gene networks. Integrated efforts
for modeling and fine-tuning synthetic gene circuits are useful for
ensuring that assembled networks operate as intended. Such approaches
will be increasingly important as more complex circuits are constructed
along with the expanded development of interoperable parts. Although
studies have shown that in some cases, component properties alone
are sufficient for predicting network behavior
22,31,46
, others have dem-
onstrated the need for modeling and fine-tuning networks after their
basic topologies have been established
1,22
. A multi-step design cycle that
involves creating diverse component libraries, constructing, character-
izing and modeling representative network topologies, and assembling
and fine-tuning desired circuits, followed by subsequent refinement
cycles
22
, will be crucial for the successful design and construction of
next-generation synthetic gene networks.
The fine-tuning of biomolecular parts and networks can be
achieved by developing diverse component libraries through muta-
genesis followed by in-depth characterization and modeling
22,47–51
.
Significant progress has been made in tuning gene expression by
altering transcriptional, translational and degradation activities. For
example, promoter libraries with a range of transcriptional activities
can be created and characterized, plugged into in silico models and
then used to develop synthetic gene networks with defined outputs,
without significant post-hoc adjustments
22,47–51
. Alternatively, syn-
thetic ribosome binding site (RBS) sequences can be used to optimize
protein expression levels. Recently, Salis et al.
52
have developed a
thermodynamic model for predicting the relative translational ini-
tiation rates for a protein with different upstream RBS sequences,
a model that can also be used to rationally forward-engineer RBS
sequences to give desired protein expression. In addition, protein
degradation can be controlled by tagging proteins with degradation-
targeting peptides that impart different degradation dynamics
53
.
By automating the construction and characterization of biomo-
lecular components, extensive libraries could be created for the rapid
design and construction of complex gene networks. These efforts,
coupled with in silico modeling, would serve to fast-track synthetic
biology (more detailed discussions of modeling techniques for syn-
thetic biology are found in refs. 22,31,54–57). However, to build
reliable models of biomolecular parts and networks, new methods
the behavior of synthetic circuits; third, developing probes for reliably
quantifying state values for synthetic (and natural) biomolecular sys-
tems; and fourth, creating test platforms for characterizing component
interactions within engineered gene networks, designing gene circuits
with increasing complexity and developing complex circuits for use
in higher organisms. These advances will allow synthetic biologists to
realize higher-order networks with desired functionalities for satisfying
real-world applications.
Interoperable parts and modules for synthetic gene networks.
Although there has been no shortage of novel circuit topologies
to construct, limitations in the number of interoperable and well-
characterized parts have constrained the development of more com-
plex biological systems
22,31,35,36
. The situation is complicated by
the fact that many potential interactions between biological parts,
which are derived from a variety of sources within different cellular
backgrounds, are not well understood or characterized. As a result,
the majority of synthetic circuits are still constructed ad hoc from a
small number of commonly used components (e.g., LacI, TetR and
lambda repressor proteins and regulated promoters) with a signifi-
cant amount of trial and error. There is a pressing need to expand
the synthetic biology toolkit of available parts and modules. Because
physical interconnections cannot be made in biological systems to
the same extent as electrical and mechanical systems, interoperability
must be derived from chemical specificity between parts and their
desired targets. This limits our ability to construct truly modular parts
and highlights the need for rigorous characterization of component
interactions so that detrimental interactions can be minimized and
factored into computational models.
Engineered zinc fingers constitute a flexible system for targeting spe-
cific DNA sequences, one which could significantly expand the available
synthetic biology toolkit for performing targeted recombination, con-
trolling transcriptional activity and making circuit interconnections.
Zinc-finger technology has primarily been used to design zinc-finger
nucleases that generate targeted double-strand breaks for genomic
modifications
37
. These engineered nucleases may be used to enhance
recombination in large-scale genome engineering techniques
34
. A sec-
ond and potentially very promising use of engineered zinc fingers is
as a source of interoperable transcription factors, which would greatly
expand the current and limited repertoire of useful activators and
repressors. In fact, zinc fingers have already been harnessed to create
artificial transcription factors by fusing zinc-finger proteins with acti-
vation or repression domains
38,39
. Libraries of externally controllable
transcriptional activators or repressors could be created by engineer-
ing protein or RNA ligand-responsive regulators, which control the
transcription or translation of zinc finger–based artificial transcription
factors themselves
18
. These libraries would enable the construction of
basic circuits, such as genetic switches
1
, as well as more complex gene
networks. In fact, several of the higher-order networks we describe
below rely on having multiple reliable and interoperable transcriptional
activators and repressors for proper functioning.
Even so, these engineered transcription factors have not yet been
fully characterized, and if they are to be used as building blocks for
complex gene networks, then knowledge of their in vivo kinetics and
input-output transfer functions would be beneficial. In addition, much
of the rich dynamics associated with small, synthetic gene networks is
attributable to the cooperative binding or multimerization of transcrip-
tion factors, and it is not yet clear what further engineering is required
to endow zinc-finger transcription factors with such features.
Nucleic acid–based parts, such as RNAs, are also promising can-
didates for libraries of interoperable parts because they can be
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