www.landesbioscience.com Bioengineered Bugs 1
Bioengineered Bugs 1:6, 1-7; November/December 2010; 2010 Landes Bioscience
interoperable memory devices,
27
coun-
ters,
27
sensors,
28,29
and protein scaffolds.
30

Using these circuits, biological engineers
have created synthetic organisms that
can be used for bioremediation, biosens-
ing, computation, bioenergy and medi-
cal therapeutics (reviewed in ref. 31–33).
Despite these advances, the realization of
synthetic-biology-based applications will
require future breakthroughs in our abil-
ity to create sufficiently complex and reli-
able biological systems. Here, I will discuss
current limitations and potential solutions
for the construction, probing, modulation
and debugging of scalable biological sys-
tems as well as hurdles for the deployment
of engineered organisms from bacteria to
mammalian cells which adds to the dis-
cussion of next-generation synthetic gene
networks in reference 31 (Fig. 1).
Physical Construction of Scalable
Biological Systems
Construction of early synthetic circuits
largely relied on restriction enzymes and
polymerase chain reaction (PCR)-based
techniques to assemble existing genetic
components. These methods do not scale
well with increasing complexity due to
a lack of sufficient unique restriction
sites and the need to have physical DNA
templates from which to amplify genetic
parts. Standards for library construction
and the assembly of parts libraries
34
have
been integral in circumventing this depen-
dency on templates and restriction sites.
However, since these parts must be devoid
of restriction sites used in the defined
standards and should ideally be optimized
for use in one’s organism of choice,
35
the
use of whole-gene DNA synthesis is on
COMMENTARY COMMENTARY
Original article: Lu TK, Khalil AS, Collins JJ. “Next-
Generation Synthetic Gene Networks.” Nature
Biotechnology 2009; 27:1139–50; (http://www.
nature.com/nbt/journal/v27/n12/abs/nbt.1591.
html).
Key words: synthetic biology, biological
circuits, engineered organisms, model-
ling, high-throughput design, regula-
tory issues, biological probes, biological
modulators
Abbreviations: PCR, polymerase chain
reaction; DNA, deoxyribonucleic acid;
RNA, ribonucleic acid; Mbp, mega-
basepairs; Kbp, kilo-basepairs; qRT-
PCR, quantitative reverse-transcriptase
PCR; FRET, fluorescence resonance
energy transfer; IPTG, isopropyl β-D-1-
thiogalactopyranoside; SELEX, system-
atic evolution of ligands by exponential
enrichment; RNAi, RNA interference;
GMOs, genetically modified organisms
DOI#: 10.4161/bbug.1.6.13086
Submitted: 06/24/10
Revised: 07/19/10
Accepted: 07/20/10
Previously published online:
www.landesbioscience.com/journals/
biobugs/article/13086
Correspondence to: Timothy K. Lu;
Email: timlu@mit.edu
S
ynthetic biology is focused on engi-
neering biological organisms to study
natural systems and to provide new solu-
tions for pressing medical, industrial
and environmental problems. At the
core of engineered organisms are syn-
thetic biological circuits that execute the
tasks of sensing inputs, processing logic
and performing output functions. In
the last decade, significant progress has
been made in developing basic designs
for a wide range of biological circuits
in bacteria, yeast and mammalian sys-
tems. However, significant challenges in
the construction, probing, modulation
and debugging of synthetic biological
systems must be addressed in order to
achieve scalable higher-complexity bio-
logical circuits. Furthermore, concomi-
tant efforts to evaluate the safety and
biocontainment of engineered organ-
isms and address public and regulatory
concerns will be necessary to ensure that
technological advances are translated
into real-world solutions.
In the last century, scientists have made
giant strides in identifying and study-
ing biological parts such as proteins and
nucleic acids,
1-5
understanding regulatory
networks,
6
and constructing engineered
organisms using the ever-advancing tools
of genetic engineering.
7
In the last decade,
synthetic biologists have leveraged the
power of modern molecular biology using
frameworks translated from traditional
disciplines such as electrical engineering,
computer science, mechanical engineer-
ing and chemical engineering to create a
wide range of synthetic biological circuits,
including switches,
8-15
oscillators,
16-18
digi-
tal logic gates,
19-23
filters,
24-26
modular and
Engineering scalable biological systems
Timothy K. Lu
1,2
1
Synthetic Biology Group; Research Lab of Electronics; Department of Electrical Engineering and Computer Science; Massachusetts Institute of Technology; and
2
Broad Institute of MIT and Harvard; Cambridge, MA USA
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2 Bioengineered Bugs Volume 1 Issue 6
what DNA to write (Fig. 2). Just as the
decoding of the human genome sequence
did not immediately reveal the functions
of all human genes, the utility of high-
throughput DNA synthesis technology
will only gradually become evident as
synthetic biologists learn how to create
complex systems. For example, future
synthesized circuits should be designed
with ease of probing, modulating and
debugging in mind. These features could
be implemented by including validated
RNA “handles” that can be easily mea-
sured with standard probe sets to deter-
mine internal RNA concentrations, gene
circuits that allow inducers to modulate
synthetic circuit protein levels, and prop-
erly situated restriction sites for the rapid
cloning of components that need system-
atic optimization, such as ribosome bind-
ing sequences.
Significant advances in well-charac-
terized, interoperable devices are neces-
sary for the construction of higher-order
modules that will enable scalable biologi-
cal systems.
38
The majority of biological
circuits have been constructed using a
handful of synthetic parts.
31
Furthermore,
it is often the case that when new designs
for biological parts are developed, only a
few instantiations are created and tested,
usually in single cellular backgrounds. As
a result, there is a need for the systematic
DNA synthesis productivity has exceeded
1 Mbp per person per day while Venter
and colleagues recently succeeded in syn-
thesizing a 1.08 Mbp genome.
37
However,
most synthetic gene circuits to date have
not exceeded the 50 Kbp level, indicating
that there is a large gap between our abil-
ity to read and write DNA and knowing
the rise.
36
Using direct chemical synthe-
sis, circuits can be designed in silico and
implemented in DNA with significantly
less effort from researchers. As DNA syn-
thesis becomes increasingly economical
and efficient, it will become possible to
construct complex systems with less reli-
ance on restriction enzymes. For example,
Figure 1. A basic design cycle for synthetic biology includes creating well-characterized parts (e.g., regulatory elements, genes, proteins, RNAs), con-
structing synthetic devices and modules and designing and assembling higher-order networks. All steps of this cycle are aided by modelling, probes
and modulators to analyze circuit performance. Debugging is an iterative process based on parts optimization, ne-tuning regulatory components,
modelling and changing circuit architecture.
Figure 2. DNA sequencing and synthesis technologies are advancing at exponential rates,
outpacing the ability of synthetic biologists to construct useful and scalable biological circuits.
36

These trends are similar to Moore’s law for integrated circuits
72
and suggest that there is substan-
tial room for growth in the eld of synthetic circuits.