Alterar Fuerza Promotor

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Tuning Response Curves for Synthetic Biology

Jordan Ang, Edouard Harris, Brendan J. Hussey, Richard Kil, and David R. McMillen*
Department of Chemical and Physical Sciences and Institute for Optical Sciences, University of Toronto, Mississauga, Ontario,
Canada L5L 1C6
ABSTRACT: Synthetic biology may be viewed as an effort to

establish, formalize, and develop an engineering discipline in
the context of biological systems. The ability to tune the
properties of individual components is central to the process of
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system design in all fields of engineering, and synthetic biology

is no exception. A large and growing number of approaches
have been developed for tuning the responses of cellular
systems, and here we address specifically the issue of tuning
the rate of response of a system: given a system where an input
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affects the rate of change of an output, how can the shape of the response curve be altered experimentally? This affects a system’s
dynamics as well as its steady-state properties, both of which are critical in the design of systems in synthetic biology, particularly
those with multiple components. We begin by reviewing a mathematical formulation that captures a broad class of biological
response curves and use this to define a standard set of varieties of tuning: vertical shifting, horizontal scaling, and the like. We
then survey the experimental literature, classifying the results into our defined categories, and organizing them by regulatory
level: transcriptional, post-transcriptional, and post-translational.
KEYWORDS: synthetic biology, tuning, tunable, response curves, biological rates
S ynthetic biology includes a concerted effort to formalize an

engineering discipline suitable for the design and
implementation of novel biological systems.1−8 Analogies to
Synthetic biology will require these same tuning capabilities,
for the same reasons: if we are to build complex systems in
biology, we must be able to tune both the internal dynamics of
well-established fields such as mechanical or electrical engineer- individual systems and to match the output/input levels of
ing are often drawn, but it has also been noted9 that biology connected systems. A growing library of experimental work has
presents a number of particular challenges for engineering demonstrated the ability to tune biological response curves, and
applications: the biological environment is noisy, our under- here we will review a number of approaches that have been
standing of cellular dynamics is imperfect, and our tools for implemented in vivo in a variety of biological contexts. After an
creating and manipulating biological systems are limited and introduction to the mathematical description of response
still under active development. Engineering in a cell is currently, curves, we will group our discussion into sections on
and perhaps in some ways fundamentally, more difficult than transcriptional, post-transcriptional, and post-translational
engineering in steel or silicon. levels of regulation. It is a positive sign for the future progress
One advantage offered by the advanced state of development of synthetic biology that there are now so many publications on
in other branches of engineering is the ability to tune the way this topic, but it also means that we cannot claim that this
review is exhaustive.
■
individual components respond to their inputs. Let us
introduce the generic idea of a process: a system that accepts
an input (mechanical force, electrical current, or biomolecular RESPONSE CURVES: MODELS AND MECHANISMS
concentration) and responds dynamically by changing its We begin by establishing a mathematical notation to be used
output (bending, current flow, or concentration of another throughout the remainder of the review. Because tuning can
biomolecule) at some predictable rate. We will refer to the take many forms, we want our description to be as broadly
relationship between the input to a process and the rate at applicable as possible. We confine ourselves to population-
which the process changes its output as a response curve: a averaged quantities, without addressing the range of single-cell
mapping from input levels to output rates of change. distributions that can generate a given population-level
Mechanical and electrical engineering projects have an response. Average rates of response in a biochemical system
extensive ability to tune these response curves: a girder can can often be represented by curves that rise steadily from a
flex at a desired rate or resonate at a desired frequency; a circuit minimum rate to asymptotically approach some maximum rate
element can slew its current or voltage output rapidly or as the input is increased (for activating inputs), or fall steadily
gradually. This tunability allows engineers the powerful abilities from a maximum to a minimum rate (for repressing inputs)
to design individual components with desired behaviors and to
integrate multiple components by ensuring that inputs and Received: May 15, 2013
outputs match across different processes. Published: August 1, 2013
© 2013 American Chemical Society 547 dx.doi.org/10.1021/sb4000564 | ACS Synth. Biol. 2013, 2, 547−567
ACS Synthetic Biology Review
Figure 1. Basic ways in which to transform the shape of sigmoidal response curves. Dark curves are reference curves; light curves are altered curves.
Also shown is a Hill function representation for each of the curves; parameters responsible for each of the transformations are bolded. Note that
these transformations are not linearly independent. In order to affect only the leakage level in panel E, k′ and k must be tuned in opposite directions
such that their sum remains constant. Experimental methods for achieving these transformations are discussed in the main text.
(see Figure 1A). Nonlinear, monotonically increasing curves of dy

this general type can describe Michaelis-Menten kinetics for = f (x )
dt (1)
enzyme-catalyzed reactions, the rate of transcription from a
promoter as a function of an activating or repressing where x and y are the input and output of the process,
transcription factor protein, and a variety of other examples. respectively, and f(x) defines the response curve. Strictly
The frequent appearance of these saturating response curves in speaking, such equations arise only from elementary chemical
biology arises because many in vivo biochemical reactions are reactions or from multistep reaction systems where a strong
rate-limited by the concentration of some conserved macro- separation of time-scales yields a single rate-limiting step. In
molecule (such as DNA or an enzyme). The specific shapes of many situations, however, it is possible to approximate more
these curves are governed by the details of individual systems, complex systems with simplified first-order systems, often
and parameter changes lead to a range of alterations (see informed by empirical observations of the system in question;
Figures 1B−G). this approach finds common use, and we will adopt it here.
We will focus on biological processes whose response curves The Hill function10,11 provides a semiempirical approach
can be described (or well approximated) by a single first order capable of capturing the class of response curves of interest.
differential equation of the form The function describes the average fraction of binding sites (of
548 dx.doi.org/10.1021/sb4000564 | ACS Synth. Biol. 2013, 2, 547−567
Figure 2. Simplified view of the biochemical mechanisms behind regulated transcription and their relation to tuning the rate response curve for gene
expression. The input and output signals are the molecular concentrations of a transcription factor protein (TF) and an expressed protein,
respectively. RNAP = RNA polymerase, O = operator site (TF binding sequence), TATA = RNAP binding sequence (TATA box in eukaryotes and
archaea, −10 and −35 consensus sequences in bacteria), RBS = ribosome binding site. (Top) Transcriptional activation where the promoter-bound
TFs promote the recruitment of RNAPs, increasing the probability per unit time that a RNAP will bind. Tuning parameters are in reference to eq 3.
(Bottom) Transcriptional repression via steric inhibition, wherein one or more TFs physically block RNAP binding to the promoter or impede its
progress along the template DNA strand (the latter case is illustrated here). Tuning parameters are in reference to eq 4. In general, mechanisms for
both activation and repression vary31 and can involve more complex actions including altering DNA secondary structure and recruiting additional
coregulator proteins; in eukaryotes, RNAP binding is mediated by a suite of accessory proteins.
some biomacromolecule, say) occupied by an input ligand, as a function term used to describe the up- or down-regulation of
function of unbound ligand concentration, x: gene expression by a regulatory species. For activation, the
expression rate increases by an additional amount proportional
xn to θ (where x is the concentration of the regulatory species),
θ (x ) =
K + xn
n (2) such that the rate of new protein production may be described
by
with the parameters K and n described below. It has a sigmoidal
shape, ranging between 0 and 1 as x increases; the approach to
1 represents saturation, where binding sites are nearly fully
occupied at all times. where y is the concentration of the protein being expressed, k′
The K parameter (the Hill constant) is related to the is the basal rate of production, k is the maximum additional
dissociation constant between the ligand and the macro- production rate arising from up-regulation, and the bracketed
molecule: it is equal to the ligand concentration for which half term is an increasing sigmoidal Hill function. Repression, on
of all the possible binding sites become occupied. It therefore the other hand, may be modeled by replacing the regulated
also serves as a rough indicator of the level of ligand production term with k(1 − θ) such that
concentrations needed to induce saturation (x ≫ K).
In some cases, if a macromolecule is already bound by a
ligand, the binding affinity of subsequent ligands to that
macromolecule becomes enhanced or reduced; this is known as Here, k′ + k is the basal (unregulated) expression rate, k′
cooperative binding, quantified in the Hill function by n (the accounts for the fact that complete repression may not be
Hill coefficient). n = 1 indicates a noncooperative reaction; n > possible, and the bracketed term is a decreasing sigmoidal Hill
1 indicates cooperativity, where affinity increases in the function.
presence of previously bound ligands; and 0 < n < 1 indicates This type of description greatly abstracts the realities of
negative cooperativity, where affinity is reduced. The larger the biological processes: in addition to combining multistep
value of n, the steeper the slope of the Hill function. processes such as gene expression into a single step, it neglects
Synthetic biologists often take advantage of time-scale fluctuations, assuming instead that a population-averaged view
separations or leverage longer time-scales of interest to model will be sufficient for at least any initial design work. In cases
multistep processes as single-step events using an empirically where these realities cannot be neglected, these simplifications
parametrized Hill function. The modeling of gene expression will need to be reconsidered.
that is transcriptionally or translationally activated or repressed Rates of change tend to be difficult to measure
by an input signal (see Figure 2) commonly follows this experimentally. Consequently, reports of steady-state input-
practice, since the shape of the Hill function has been shown to output (or “dose-response”) functions are seen in literature far
agree well with experimental evidence.12 In such cases, the rate more often than the rate response curves we describe above.
of change of protein concentration may be described by Fortunately, it is possible to interconvert the two if the ancillary
combining basal (unregulated) gene production with a Hill processes contributing to steady state are well-known. Consider
a biochemical process described by eq 1, where the output y one whose binding helped to recruit RNA polymerase (RNAP),
represents the concentration of a protein whose production then this could be achieved by tuning the activation potency of
rate is given by the response curve f(x). The total intracellular each bound TF protein, i.e., changing the probability per unit
protein concentration, ytot, depends both on the protein’s rate time that an RNAP will bind per bound TF. For down-
of production and its rate of removal; in the simplest case, this regulated expression, we are not aware of any single-step
can be represented by a linear degradation term in the rate method of transforming the response curve in this manner
equation for ytot: without also affecting the final leakage level; such tuning would
dytot likely require combining other transformations. For example, in
= f (x) − kdytot the special case where k′ = 0, vertical extension becomes
dt (5) identical to vertical scaling, and a supplementary vertical shift
where kd is a first-order rate constant. Setting eq 5 to zero, we could be used to adjust the baseline level to a nonzero value.
can easily extract the steady-state relationship for total Leakage. We refer to the low-end level of the response
intracellular y as a function of constant-valued x: ytot,ss = curves as the output leakage level. It represents the portion of
f(xss)/kd (the ss subscript denotes steady-state). This is simply the process that is always activated in the up-regulating case,
the original process’s rate function scaled by kd, implying that and the portion that cannot be repressed in the down-
both the production rate and steady-state response curves share regulating case. Tuning this level without affecting the high-end
the same characteristic shape governed by the same biological saturation level (Figure 1E) is equivalent to tuning both k′ and
parameters. More generally, the degradation rate can be k, while keeping their sum k′ + k constant. For down-regulated
nonlinear, but it remains possible to extract the f(x) rate gene expression, this would amount to varying the repression
response curve. strength of each bound TF protein. In the up-regulated case, we
There are several motivations for tuning response curves in are not aware of a direct method to produce such tuning;
synthetic biology design work. When creating an analog control achieving the effect would likely require combining other
system,13−21 the nature of the response curve is critical in transformations.
determining the feedback properties of the controller. More Horizontal Scaling. Horizontal scaling, illustrated in Figure
generally, the intersections and slopes of response curves 1F, results from tuning of the Hill constant K (increasing K
determine the locations and stability properties of steady states, scales the curve to the right), which is related to the effective
and controlling steady-state positions is required for virtually binding affinity of the input signal to the process. For
any multicomponent engineered system; this includes digital transcriptionally regulated gene expression, this corresponds
logic systems, where steady states determine the values and to tuning the binding affinity of the TF to the promoter.
degree of separation of the digital ON and OFF states.22−26 Steepness. Changes in curve steepness are shown in Figure
Types of Tuning. Let us examine how each of the 1G and result from tuning the Hill coefficient n (increasing n
transformation types depicted in Figure 1 relates in principle to leading to increasing steepness). Having a steep or switch-like
the Hill function description and to underlying biomolecular steady-state response curve is often referred to as having
interactions. To make the discussion more concrete, we will ultrasensitivity in a biochemical process.27−30 Biochemically,
continue to use transcriptionally regulated gene expression changing steepness requires adjusting the effective binding
(Figure 2) as a running example; any tuning mechanisms cooperativity. For transcriptionally regulated gene expression,
mentioned in this context will be further expanded upon in the this implies the cooperative binding of multiple TFs to the
next section. Recall that for this particular process, the input same promoter (or another biochemical process that mimics
and output signals are molecular concentrations of the this effect).
transcription factor (TF) protein and the expressed protein, Dynamic Range. Often times, published experimental
respectively. results report only values at the extremes of a biological
Vertical Scaling. In Figure 1B, the response curves are scaled response curve (uninduced and fully saturated induction) or in
vertically, amounting to multiplication of the function f(x) (i.e., some instances just the ratios of the saturated levels in the form
equal scaling of k′ and k in our Hill function representation). of “fold increases”. In such cases, the precise nature of the
Most directly, this is done by creating multiple replicates of the tuning can be ambiguous. Where possible, we speculate on
entire process. For the gene expression example, this is plausible tuning effects for the full response curve; however, if
analogous to changing the promoter-gene copy number. this is not possible, we simply refer to the observed tuning as a
Alternatively, altering translational efficiency by modifying the change in the response curve’s “dynamic range”, which in
RBS strength or through codon optimization would also reality can be achieved in many ways, particularly through
vertically scale the response curve. vertical scaling, vertical extension, leakage tuning, or combina-
Vertical Shifting. Figure 1C shows the response curve tions thereof.
shifting vertically, corresponding to a change in k′ alone. This Note that all of the above descriptions assume that the
could be achieved by introducing or tuning a constitutive output is not subject to biological limitations beyond those
source of y output (i.e., one that is not regulated by the input imposed by the input signal itself, but if this is not the case, it
signal x). In the gene expression example, such an output could change the nature of the apparent tuning. As an example,
source would amount to gene transcription from a constitutive the output of an activated process could hit an absolute
promoter, supplementing transcription from the x-regulated maximum rate for the cell, perhaps because of limitations in the
promoter. availability of a substrate (e.g., nucleotides or amino acids) or a
Vertical Extension. Figure 1D shows a transformation type facilitating enzyme shared among other processes (e.g.,
that we have termed vertical extension, where the curve is polymerases or ribosomes). In this case, changes that would
scaled vertically but with the low end level fixed. Such a normally result in vertical shifting could manifest instead as
transformation would result from changing k alone. If gene leakage, as the high end of the curve ran into the upper limit of
expression were up-regulated by an activating TF, presumably attainable rates while the low end continued to shift up and
Figure 3. Examples of experimental tuning curves for transcriptional regulation. (A) Vertical scaling (left) and leakage tuning (right) on log-scale
plots, both achieved by varying the position of a promoter relative to a bacterial genome’s origin of replication.32 The gray and green curves represent
operons nearest and furthest from the origin of replication, respectively. Image used by permission of Oxford University Press. (B) Tuning of
steepness and leakage, by varying the position of operator sites relative to the TATA box.33 Copyright 2007 National Academy of Sciences of the
United States of America.
down. We assume in what follows that such global saturation is Gene Copy Number and Location. Consider an operon
not at work in the systems discussed, but the possibility should consisting of a gene under the control of a TF-regulated
be recognized. promoter. The expression response curve for the gene could be
■
vertically shifted upward by introducing additional copies of
BIOLOGICAL OPTIONS FOR TUNING the gene under the control of a constitutive (unregulated)
promoter, as this would contribute a flat baseline expression
We now survey some specific examples of response curve
rate. On the other hand, increasing the copy number of the full
tuning from the experimental literature, grouped by the
operon would act as a multiplier for the rate at which mRNA is
biological levels at which they operate: at the transcriptional
level, through post-transcriptional effects, or at the post- produced and therefore translated, leading to vertical scaling of
translational level. All input and output values will refer to the original expression response curve. This can be
molecular concentrations and rates of concentration change, accomplished by inserting multiple repeats of an operon into
respectively, unless otherwise noted. Note that while the curves the genome34 or by carrying the operon on plasmids, where
in Figure 1 provide a useful framework for discussing types of copy number is variable. Plasmid copy number is typically
tuning, experimental results are of course rarely so clean. controlled by changing the plasmid’s origin of replication,
Beyond the inevitable experimental noise, it is often the case although Chen et al.35 showed that the copy number of widely
that secondary biochemical effects lead to secondary tuning used 2-μm-based plasmids inserted into yeast can be increased
effects. by decreasing the output and stability of a selective marker gene
Transcription. The stability of DNA, the wide array of produced by that plasmid. A library of plasmids combining
molecular biology techniques available for its manipulation, and these two effects exhibited up to a 3-fold increase of a
the generally modular structure of an operon have combined to constitutively expressed reporter gene from the same plasmid,
make transcriptional regulation a natural first target in the indicating an increase in copy number.
development of synthetic biology. Methods for controlling and A recent study by Block et al.32 has demonstrated that the
tuning gene expression via transcription act predominantly proximity of the output protein operon or the TF expressing
through mutations to operons and transcription factors (TFs); operon to the origin of replication of a bacterial chromosome
most often, process inputs in this section will be TF can vertically scale or affect the leakage level, respectively, of
concentrations. the corresponding expression response curve; see Figure 3A. In
a related study, Bikard et al.36 shuffled the order of genes in a Operator Site Modification. Modifying the sequence,
polycistronic operon encoding tryptophan production to number, or position of operator sites within a promoter are
increase the dynamic range between saturated expression common tuning techniques in synthetic biology. Since
levels as high as 11-fold over the native arrangement. These sequence modification will likely affect TF-promoter binding
efforts demonstrate that tuning is possible solely by controlling affinity, horizontal scaling of the TF vs expression response
genetic context. curve can be expected. Adding multiple copies of an operator
Promoter Modifications. Regulating gene expression by site will permit multiple TFs to bind to a single promoter and
controlling the promoter region of an operon dominated early may therefore increase maximal activation or repression levels,
work in genetic control and remains a primary technique today. leading to upward vertical extension or less leakage,
Promoters are modular genetic units that often function across respectively. It should also result in outward horizontal scaling,
entire kingdoms, and the wide range of well-known native and since the number of potential TF binding locations is greater,
synthetically designed promoters across a variety of species thereby requiring a higher TF concentration to reach binding
offers choices for expression ranges; the Registry of Standard saturation. Furthermore, if TF binding to adjacent operator
Biological Parts (http://partsregistry.org) provides a conven- sites is cooperative, then response curve steepness would also
ient catalog of available and pretested options. Promoters are be affected. Finally, we would expect operator site location to
increasingly being characterized under similar genetic con- affect the ability of a TF to recruit or hinder the binding of
ditions for comparisons of strength.37−39 RNAPs, therefore leading to changes in vertical extension or
While earlier studies developed our understanding of the leakage, respectively. In practice, however, tuning results are
nucleotide architecture of promoters and the correlated rarely so straightforward. Moving operator site position, for
mechanisms by which promoters function (reviewed in refs example, could lead to significant changes in the secondary
40 and 41), many recent investigations have focused on the structure of the DNA, TF binding notwithstanding, and
tunable nature of this relationship. Particular targets for tuning therefore lead to additional vertical scaling effects.
have been the binding strengths of communal proteins that Murphy et al.33 (and an early study by Heins et al.53 that did
make up the general transcriptional machinery (e.g., RNAP and not report full response curves) used operator site modification
sigma factors), which we focus on first, and the binding in S. cerevisiae, varying the number of operator sites binding the
strengths and activities of TFs, which we cover in the TF repressor TetR, and their proximity to the TATA box, to
subsequent section on operator site modification. obtain a variety of expression response curves with differences
The binding sites for the RNAPs, the TATA box for in vertical scaling, steepness, and most prominently, leakage
eukaryotes, and the −10 and −35 hexameric upstream regions levels (ranging from approximately 0.2% to 35% of the
in prokaryotes, are a set of consensus sequences that vary across unrepressed output). A sampling of these observed curves is
the kingdoms in which they are found. An example of the shown in Figure 3B. The experimental response was measured
sequence dependence of the binding strength was demon- as a function of the chemical inducer anhydrotetracycline
strated by Eandwar et al.,42 who showed that mutations of the (aTc), which acts to reduce the binding of constitutively
binding region of the T7 promoter, targeted by the T7 RNAP expressed TetR to the operator site(s), and the curves thus
(originally from T7 bacteriophage but used widely as a show activation as a function of increasing aTc; if measure-
heterologous RNAP in bacteria and eukaryotes), reduced the ments were taken while varying the concentration of TetR
binding affinity 2- to 3-fold. In principle, this would directly, we would expect a decreasing response curve
horizontally scale outward the T7 RNAP vs expression representing repression.
response curve and vertically scale downward any TF vs With regards to TF effectiveness, many operator sites show
expression response curve. The precision with which this is optimal proximities from the RNAP binding sites.54,55 For
possible has been aided by more recent efforts to create repressors, the mechanism by which the TF prevents the
libraries of TATA box43,44 and hexameric sequence45,46 binding of the polymerase to the DNA likely determines the
mutations. influence of operator position. A recent study by Garcia et al.55
Since the discovery of the consensus sequences, mutations in has suggested that the mechanism of repression by the Lac
the surrounding regions have also been known to influence the repressor is different for operator positions centered at −60 vs
output of gene expression,47,48 with vertical scaling of the +11, resulting in differences in leakage.
expression response curve over as much as 3−4 orders of Libraries of operator site and TF mutants are also increasing
magnitude.49 in number. These typically target the binding affinities of the
Upstream sequence (UP-element) interactions with the C- operator-TF pairs and are similar in their goals and methods to
terminal domains of the RNAP have also become a target for promoter libraries.56 Milk et al.57 combined a library of
controlling gene expression.50 A recent study by Rhodius et mutations in the Lac repressor at three residues known to
al.51 determined the upstream contributions to promoter interact with the operator, with a library of symmetric
strength using a library of 60 mutated promoters. This library mutations in the Lac operator at bases 5 to 7, to produce a
included mutations distal to the −35 hexamer, as far as −65 bp range of repression options spanning a 35-fold difference in
upstream. Different mutations of the UP-elements led to leakage. Maity et al.58 found that single-nucleotide changes to
vertical scaling, achieving 2-fold increases and 4-fold decreases lac O1, the primary operator of the E. coli TF repressor LacI, led
in gene expression. These findings were then modeled along to changes of up to 6- and 12-fold in repressed and
with mutations in the core promoter regions to include all the nonrepressed expression levels, respectively, indicating a
DNA elements that contribute to promoter strength. In a study combination of tuning types at work.
of over 2,800 constructs in yeast, Gertz et al.52 assembled a Promoter Escape. While the tuning approaches discussed
library of enhancers with random combinations of operator thus far focus on regulating transcription initiation (RNAP
sites upstream from a promoter, offering a finely tuned range of binding), progress has also been made concerning the
basal expression. regulation of promoter escape: the ability of the transcriptional
complex to dissociate itself from the promoter and allow principle, the use and variation of TSDs would vary the leakage
elongation of the full transcript, the failure of which leads to level in the expression response curve.
abortive transcripts. The 20-nucleotide sequence directly TAL-Effectors and Zinc Fingers. While importing heterol-
downstream of the transcription start site can have a dramatic ogous TFs into a system of interest has been a productive
influence on the efficiency of promoter escape. Kammerer et strategy, there are limits to both how many such TFs are
al.59 showed that the bacteriophage T5 N25 promoter and its currently available and the degree of orthogonality achievable
derivative, the N25 antipromoter, exhibit very different rates of between them. These limitations have inspired the creation of
promoter escape (roughly 1.7 and 0.6 min−1, respectively) and synthetic TFs, constructed by fusing together TADs with
ratios of abortive to productive transcripts (40 and 300, protein domains engineered to bind particular DNA sequences
respectively), despite differing only in the initial portion of their with high specificity. These synthetic TFs have introduced the
transcribed sequences (+3 to +20). Chander et al. 60 ability to activate gene expression in eukaryotes without the
demonstrated finer tuning using individual mutations to the need for either native or heterologous promoter-TF pairs.
20-nucleotide sequence. These changes should in principle Transcription activator-like effectors (TALEs), first discov-
introduce a vertical scaling of the expression response curve. ered in the Xanthomonus genus, have recently become an
Using a library of 43 variants and a highly abortive promoter, important focus of the synthetic TF field. TALEs comprise
Hsu et al.61 demonstrated a 25-fold range of promoter escape tandem repeats of small 33−35 amino acid domains, each of
efficiency in vitro, resulting in an mRNA increase ranging from which recognizes and binds a single nucleotide. Covalent
5% to 150% above the native level, and vertical scaling of the linkage of these domains into engineered arrays allows for the
rate of gene expression. Manipulation of promoter escape highly specific recognition and targeting of longer, user-
efficiency has since been demonstrated in vivo in E. coli,62 specified nucleotide sequences.74 In a recent study targeting
suggesting a key gene expression tuning approach for operons regions in a DNase I hypersensitive site in human HEK293T
where promoter escape is rate limiting. cells, Maeder et al.75 created TALEs with varying numbers of
Modular Transcription Factor Domains. Typical eukaryotic domain repeats, allowing them to incrementally tune the
TFs have a modular structure comprising two to three dynamic range of expression between 5.3- to 114-fold. In
domains:31 a DNA binding domain (DBD), a trans-activating addition, fusion to two distinct TADs, p65 and VP64, were
or trans-silencing domain (TAD, TSD), and an optional signal- compared. The VP64 construct yielded consistently higher
sensing domain (SSD) that affects TF activity primarily by expression, which we speculate is a result of vertically
modulating the DBD binding affinity for its cognate DNA extending the response curve.
operator sequence in a signal-dependent manner. Modularity is In another study, Perez-Pinera et al.76 engineered several
conferred by the fact that these domains typically function TALEs (using the VP64 TAD) to target various upstream
independently, allowing for the creation of chimeric TFs regions within four endogenous gene promoters (distributed
through domain mixing63,64 with tuning implications that vary within 600 bp of the transcription start site) in human
with the domains involved (see below). HEK293T cells. They observed modest transcriptional
Signal-Sensor Domains. Signal-sensor domains (SSDs) that activation when using individual TALEs, but considerable
respond to exogenous stimuli (e.g., small molecules, light, etc.) synergistic activation effects for three of the four genes when
permit externally inducible control over effective TF-promoter expressing certain combinations of TALEs, with increases in
binding affinity. In principle, this leads to horizontal shifting of mRNA abundance spanning a striking 4 orders of magnitude.
the expression response curve that corresponds to TF By systematically varying these combinations, output gene
concentration as the process input (and not the exogenous expression levels were tunable over a 500-fold range (here
signal). To date, a wide variety of eukaryotic TFs have been TALEs were constitutively expressed from a common
created by co-opting inducible DNA binding proteins from promoter, and therefore full TALE vs expression response
bacteria (see ref 65 for many examples) and inserting their curves were not reported since TALE concentrations were not
cognate operator sites into minimal promoters.66 titrated).
trans-Activator and trans-Silencer Domains. For an Similar in concept to TALEs, eukaryotic zinc fingers (ZFs)
activating TF, functionality can also be adjusted by varying are small (∼30 amino acid) modular domains that bind to 3 bp
the type and/or number of trans-activator domains (TADs). DNA regions with engineerable sequence specificity and can be
Since TADs recruit transcriptional machinery through direct linked into multifinger arrays that recognize longer sequences.77
binding interactions with coactivator proteins, varying these In recent work, Khalil et al.78 used the OPEN platform79 to
domains changes the activation potential of each individual TF, construct an artificial library of specific and orthogonal ZF
thereby in principle achieving vertical extension (adjusting the array-promoter pairs, in particular, three-finger arrays that bind
high-end saturation limit of the activation response curve). to cognate 9-bp operators. They then fused the arrays to a
Although the potent Herpes simplex VP16 TAD67,68 is most minimal VP16 TAD to create a library of synthetic TFs (ZF-
commonly used, graded regulation has been demonstrated by TFs) that activated expression with dynamic ranges ranging
fusing tandem repeats of VP16-derived minimal domains (e.g., from 1.3- to 6-fold and then showed that key TF properties
the quad-repeating VP64 TAD) and other TADs such as could be rationally and independently adjusted to further tune
human NF-κB-derived p65 and human-derived E2F4.69−71 If transcriptional output. First, they multimerized ZF-binding
cognate operator sites are cloned downstream of a constitutive operator sites in order to recruit greater numbers of ZF-TFs.
promoter, a DBD alone can function as a transcriptional For promoters harboring one, two, and eight tandem operators,
repressor through steric RNAP hindrance, although the corresponding increases in maximal expression were observed
repression potential can often be increased by fusing a trans- (indicating a possible increase in vertical extension, although
silencer domain (TSD) such as yeast-derived Ume6 or human full response curves were not presented); interestingly, a less-
kox1-derived KRAB.72,73 TSDs typically recruit corepressor and obvious decrease in leakage was also seen. They then
subsequently histone proteins that alter DNA accessibility. In performed structure-guided mutation of the ZF array backbone
(i.e., outside the DNA recognition helices) to decrease its DNA- (where the input x remains representative of total nuclear-plus-
binding affinity, presumably implying outward horizontal cytoplasmic TF concentration), thereby horizontally scaling
scaling of the expression response curve. Decreased expression the expression response curve.
levels were indeed seen as the number of mutated residues Nuclesome-Disfavoring Sequences. Recently, the use of
increased from one to four. Finally, they tested configurations nuclesome-disfavoring sequences has emerged as a promising
in which two different ZF-TFs could dimerize via the addition tuning technique in eukaryotes. TF-promoter binding is
of modular PDZ protein−protein interaction domains from regulated by nucleosomes, segments of DNA wrapped around
metazoan cells. In this case, expression from a single promoter histone proteins that are the fundamental repeating unit of
containing the two corresponding operator sites was shown to chromatin structure, that restrict access to potential operator
be synergistic in nature when both ZF-TF types were present, sites. In a recent study, Raveh-Sadka et al.88 tuned transcription
with increased vertical extension due to the interaction. activation in yeast by targeting local nucleosome organization,
In a recent study, Lohmueller et al.80 fused various leucine accomplished by the insertion of poly(dA:dT) tracts (homo-
zipper (LZ) homodimerization domains to ZF-TFs and found polymeric stretches of deoxyadenosine nucleotides, highly
that these added domains improved activation and repression prevalent in natural eukaryotic promoters and known to
up to 2.5- and 7.5-fold, respectively, using a human c-Jun LZ disfavor nucleosome formation) into a specific promoter.
(dimerization kd = 448 μM) and up to 10-fold and 8-fold, Rational fine-tuning was demonstrated by systematically
respectively, using a stronger homodimerizing yeast GCN4 LZ varying the length, composition (i.e., purity), and relative
(kd = 8 nM). This corresponds to vertical extension tuning in distance from the activating TF operator site of the inserted
the activating case and leakage tuning in the repressive case. poly(dA:dT) tract. Manipulating these tracts affects TF access
Orthogonal RNA Polymerases. RNAPs that are orthogonal to its cognate operator site, which in principle results in
to native promoters offer an alternative to activating TFs. modulation of the average TF to DNA binding affinity and,
Orthogonality permits varied concentrations within a cell therefore, horizontal scaling of the expression response curve.
without compromising native cell function and therefore varied There are two standout benefits of this technique: First, it offers
rates of transcription exclusively from cognate (RNAP-specific) much finer control over gene expression than possible even
promoters. Temme et al.81 developed a set of four orthogonal with singular point mutations to the TF operator site. Second,
variants of the heterologous T7 RNAP along with cognate it works around the problem of limited orthogonal TFs; for
polymerase-specific promoters for use in E. coli. Similar to example, poly(dA:dT) tracts can generate a multiplicity of
swapping TF-promoter pairs, these T7 RNAP variants could be responses from different promoters using the same common
interchanged to alter the expression response curve, albeit TF.
coarsely and unsystematically (published results show varying Post-Transcription. Expression control at the translational
output levels at saturating T7 RNAP concentrations, level represents a promising alternative to the control of
corresponding to varying vertical scalings or extensions, but transcription initiation. mRNA transcripts are often targeted by
omit full response curves); some finer tuning, however, was RNA-based regulators on the basis of Watson-Crick base
achieved via mutagenesis to a 5-bp strength-determining region pairing. This has enabled researchers to design and tune novel
of the promoter. regulators using model-based techniques, permitting a system-
Nuclear Localization and Export Sequences. In eukaryotic atic engineering approach not yet available for the protein
cells, genetic material and transcriptional machinery is effectors used in transcriptional control. In this section, we
contained in the nucleus, segregated from translational and present methods to tune constitutive translation rates
metabolic machinery in the cytoplasm. The bidirectional (modifying ribosome binding sites (RBSs), codons, and
translocation of TFs through the nuclear envelope (via nuclear mRNA degradation rates) and translation rates that are
pore complexes) facilitates another layer of transcriptional inducible by noncoding RNA or other small molecule effectors
regulation, as transfer rates vary among different proteins and in (riboregulators, aptamers, and RNA interference (RNAi)). For
some cases for the same protein depending on its state of post- a general review of natural RNA-based regulatory devices, see
translational modification (e.g., phosphorylation state). While ref 89; the engineering and current diversity of synthetic
macromolecules smaller than ∼40 kDa can passively diffuse devices are reviewed in refs 90 and 91, respectively.
through these pores, most proteins with intranuclear function Ribosome Binding Site Modifications. Modifying the RBS
undergo active but selective transport mediated by nuclear on an mRNA transcript alters the efficiency of translation
import and export receptors that recognize and bind to certain initiation, thereby in principle vertically scaling the overall
short amino acid nuclear localization sequences (NLSs) and expression response curve. Currently, the RBS is one of the
nuclear export sequences (NESs), respectively.82,83 Sequence most attractive options for tuning because its strength can be,
variation generates different binding affinities, which correlate in large part, forward-engineered using model-based design.
to protein import and export rates and therefore nuclear Citing the previous work of others as their foundation, Salis et
concentration.84 By fusing different NLSs and (in some cases) al.92 developed a predictive method for designing synthetic
NESs to the termini of proteins, researchers have shown the RBSs for any gene of interest based on statistical thermody-
ability to adjust the ratio of in vivo steady-state nuclear to namic modeling. Experimental validation of over 100
cytoplasmic accumulation over a wide range in different predictions in E. coli showed the method’s predictive accuracy
eukaryotic cells including yeast.84−86 Furthermore, variants of to be within a factor of 2.3 over an impressive 5 orders of
so-called classical NLSs have been generated with quantified magnitude of translational efficiency.
binding affinities to Importin (the import receptor protein) Orthogonal Ribosomes. In addition to modifying the RBS,
ranging over several orders of magnitude.84,87 Within our Hill the ribosome itself can be modified such that it becomes
function representation of gene expression, increasing the ratio orthogonal to wild-type translation, recognizing instead
of nuclear to cytoplasmic TF by some factor would be synthetic RBSs.93−95 By modifying internal segments of the
equivalent to decreasing the Hill constant, K, by the same factor orthogonal ribosome’s rRNA, horizontal scaling of translation
can be achieved.96 Through a combination of computational Practically speaking, the easiest way to obtain differing
design and experimental measurement, Chubiz and Rao97 responses using riboregulators is to simply choose from a set of
demonstrated that orthogonal ribosomes could display precharacterized heterogeneous riboregulators; if these ribor-
apparent vertical scaling by varying the sequence of the 16S egulators are functionally orthogonal, they can be used
rRNA. They further demonstrated tuning of dose-responses to simultaneously and effectively within the same cell. Isaacs et
inducers of either rRNA or cognate orthogonal ribosome al.103 produced two orthogonal crRNA-trRNA riboregulator
mRNA, achieving vertical scaling and steepness tuning.97 pairs for E. coli that exhibited 8- and 19-fold repression-to-
Start Codon Modification. While AUG is the most activation dynamic ranges, and this set was later expanded by
commonly used start codon in most species, translation can Callura et al.104 to include two additional orthogonal pairs with
also be initiated from alternative start codons that differ in ∼70- and ∼200-fold dynamic ranges. Recently, Mutalik et al.105
efficacy and differ between species. In E. coli, for example, used a model-guided design approach, involving hybridization
varying start codon usage can vertically scale an expression free energy calculations and data clustering algorithms, to
response curve, both up and down relative to the standard forward engineer new families of five and six mutually
AUG start codon.98 Similar possibilities exist in eukaryotes orthogonal trans-repressed riboregulators for E. coli with
wherein start condons seem to be especially sensitive to genetic consistent and predictable leakage levels. In the process,
context. For example, the presence and length of flanking riboregulators with leakage levels ranging from 10% to 95% of
polyU or polyA sequences can induce translation initiation the nonrepressed expression level were isolated.
from yeast non-AUG codons such as UUG, ACU, and ACG Aptamers. A post-transcriptional aptamer is a sequence of
that otherwise exhibit almost no translational activity.99 nucleotides designed, typically through an in vitro selection
Context sensitivity is also a property of prokaryote start process, to bind strongly and specifically to a ligand of
codons, where vertical scaling can arise from flanking polyA/U interest.106 Such sequences are playing an increasingly
sequences, nearby stem-loops, and variations in the proximity prominent role in post-transcriptional control devices: by
and strength of the RBS.98,100 serving as allosteric sites built into mRNA transcripts, they
Codon Optimization. In synonymous codon optimization, enable coupling between exogenous ligand concentrations and
the triplet RNA sequences coding for amino acids are replaced translation rates.107 Aptamers are normally sourced syntheti-
with alternative triplets coding for the same residue. Organisms cally: given a particular ligand of interest, the space of possible
display kinetic preference for certain codons sequences, and nucleotide sequences is methodically searched via a directed-
codon replacements can significantly affect the efficiency of evolution procedure known as SELEX (Systematic Evolution of
Ligands by EXponential enrichment).108−111
translation elongation, thereby altering translation rates and
By coupling aptamer technology to trans-acting RNA
total gene expression levels.101
molecules, Bayer and Smolke112 were able to engineer several
Welch et al.102 devised a partial least-squares based model to
ligand-controlled riboregulator systems, called antiswitches due
correlate synonymous codon choices for a particular gene with
to the fact that a ligand-induced conformational change exposes
its observed expression level in E. coli. (Interestingly, the codon
an antisense sequence (otherwise sequestered by a proximate
choices that maximized expression were not necessarily those
complementary sequence) that binds a target mRNA transcript
most commonly found in native E. coli transcripts.) Combining and blocks its translation. Tuning was demonstrated in S.
this model with a genetic algorithm allowed for the generation cerevisiae by varying the conformational equilibrium of the
of synthetic transcript sequences that produced precalculated trans-acting RNA itself: lengthening the sequestering sequence
expression levels. In principle, codon optimization allows for or introducing mismatches between it and the antisense
vertical scaling of a gene expression response curve, under the domain increased or decreased, respectively, the amount of
assumption that codon mutations do not interact with other effector ligand required to repress target mRNA repression.
control elements. This resulted in horizontal scaling and changes to the vertical
Riboregulators. A riboregulator is an RNA sequence that extension of the ligand vs expression response curve; the latter
responds to the Watson-Crick (sense-antisense) base pairing of reflecting changes to the fraction of the trans-acting RNA
a signaling nucleic acid molecule, commonly for the purpose of molecules with their antisense domains exposed in the absence
regulating translation. Isaacs et al.103 introduced a short DNA of ligands.
sequence complementary to and directly upstream from the Carothers et al.113 created a set of tunable RNA-based
RBS, such that the 5′ UTR of resulting mRNA transcripts, devices capable of delivering a wide range of gene expression
referred to as cis-repressed mRNA (crRNA), folded naturally to outputs. One set of RNA structures took the form of
form a stem-loop structure that sequestered the RBS and ribozymes: RNA structures able to catalyze reactions. These
inhibited translation initiation with extremely low leakage levels ribozymes catalyzed 5′ UTR cleavage in target mRNA, leading
(down to 2% in E. coli). Activation was then achieved by to increased mRNA half-life and thus to greater gene
independently transcribing noncoding RNA, referred to as expression. A second set of RNA structures, classified as
trans-activating RNA (taRNA), designed to target and hybridize aptazymes, exhibited similar UTR-cleavage activity but were
to the crRNA, unfold the stem-loop structure, and expose the also augmented with aptamer sequences allowing for ligand-
RBS. Tweaking sequence complementarities provided limited sensitive cleavage rates. A sophisticated modeling approach was
coarse-tuning of the taRNA vs expression rate response curve: used to guide the design process, combining a biochemical
alterations to the stem of the crRNA stem-loop structure kinetic model with RNA folding simulations. Twenty-eight
resulted in modest variations in leakage levels, while taRNA distinct RNA systems were constructed and characterized
truncation and alterations to the taRNA-crRNA hybridization experimentally, with widely varying gene expression levels
sequence influenced activation levels, possibly at least in part a suggesting ligand vs expression response curves with various
result of horizontal shifting of the response curve, since vertical scalings. Importantly, the model was successful at
variations to taRNA-crRNA binding affinity were observed. predicting the observed experimental results, and analysis of the
Figure 4. Examples of experimental tuning curves for post-transcriptional regulation. (A) Dose-response curves for IPTG-inducible pT181
transcriptional attenuators. Curves are shown for wild-type (left) and mutant (right) attenuators designed to function orthogonally. Repression
curves for a single repeat of the attenuator (circles) and for two attenuators in tandem (squares) are shown; addition of the second attenuator leads
to a decrease in leakage and an increase in steepness. Image from ref 115. Copyright 2011 National Academy of Sciences of the United States of
America. (B) Tuning of horizontal scaling and leakage of shRNA switches via modulation of the 3′ length (left) and 5′ length (right) of the region of
complementarity between the competing strand and the shRNA stem sequence of a hairpin transcript. On each plot, results are shown for multiple
sequence lengths. Image from ref 117. Reprinted by permission from Macmillan Publishers Ltd: Molecular Systems Biology, Beisel, et al., 4, 224,
copyright 2008.
model offered direct guidance in the design process by targeted methods for manipulating the rate of mRNA
identifying important steps in the biochemical kinetics and degradation.
predicting sequence mutations likely to affect those steps. This Early work examined factors involved in controlling mRNA
highlights the strong potential for tuning ribozyme/aptazyme stability, most notably the effect of hairpin secondary structures
devices through systematic, model-guided design. in the 5′ UTR of the transcript (reviewed in ref 118). By
Transcriptional Attenuators. In antisense RNA-mediated introducing rationally designed hairpins into E. coli mRNA,
transcriptional attenuation, the binding of an antisense RNA to Carrier and Keasling119 were able to influence half-lives over an
an “attenuator sequence” in the 5′ UTR of a nascent mRNA order-of-magnitude range. More recently, Babiskin and
transcript causes it to fold into a configuration that exposes an Smolke120 developed an RNA device in S. cerevisiae enabling
intrinsic transcriptional terminator hairpin, resulting in aptamer-mediated transcript cleavage. This was done by
premature transcription termination.114 Lucks et al.115 designed inserting into the 3′ UTR of the transcript of interest a
three such antisense RNA sequences to function orthogonally hairpin-shaped formation amenable to cleavage by the
in E. coli. In a method mirroring the repetition of operator sites ribonuclease Rnt1p and containing an aptamer sequence that
in a promoter, series insertion of an additional identical leads to inhibited cleavage activity when ligand-bound; in the
attenuator sequence into the 5′ UTR of the target transcript absence of ligand, Rnt1p cleavage proceeds normally and the
steepened and reduced the leakage level of the antisense vs transcript, with its polyA tail removed, is quickly degraded.
expression response curve in a manner agreeing remarkably Babiskin and Smolke120 employed three different strategies
well with the multiplication of single attenuator Hill functions to tune the monotonically increasing ligand vs gene expression
(when normalized to 1); see Figure 4A. This suggests that response curve. Changes to a key region of the hairpin
attenuators in series function independently, as in the case for sequence controlling cleavage efficiency yielded various
engineered tandem ribozyme devices.116 combinations of vertical extension and leakage tuning,
mRNA Degradation Control. The previously mentioned typically with only slight horizontal scaling. Changes to
post-transcriptional strategies affect translation efficiency per another key region of the hairpin controlling ribonuclease
mRNA transcript. An alternative approach to tuning transla- binding affinity and containing the aptamer sequence resulted
tional rates involves controlling the transcript concentrations in more notable horizontal scaling, as was expected; in
themselves; this would lead to vertical scaling of the gene addition, changes in vertical extension and leakage levels were
expression response. To this end, researchers have developed again observed, likely resulting from nucleotide modifications
to the hairpin stem that were required for variable aptamer through an aptameric distal loop embedded in the hairpin
integration. Finally, positioning multiple hairpin copies (up to sequence. The hairpin exists in equilibrium between two
three) within the 3′ UTR resulted in outward horizontal conformations, one that is efficiently processed by the RNAi
scaling and reduced leakage levels due to the increase in machinery and another that is not. In the latter, the aptamer is
potential cleavage targets for Rnt1p. In particular, the exposed and binding of a ligand stabilizes this conformation,
expression response curve’s Hill constant, K, increased nearly effectively preventing the hairpin from forming its active siRNA
additively with hairpin copy number, while the leakage level state. Thus, higher intracellular ligand concentrations reduce
decrease was approximately multiplicative (47%, 20%, and 10% silencing of the shRNA’s target protein. Aptamers responsive to
for one to three copies). hypoxanthine, tetracycline, and theophylline ligands were
RNA Interference. RNA interference is the process whereby successfully tested in human HEK293T cells. Guided by a
small double stranded RNAs (dsRNAs) down-regulate protein semiempirical thermodynamic model, the lengths of the
expression via either steric hindrance of the ribosome or complementary RNA regions in the aptamer-stabilized
induced endonuclease cleavage of the target mRNA. Both of shRNA conformations were varied, yielding systematic changes
these processes are mediated by the RNA-induced silencing to the expression response curve, primarily in horizontal
complex (RISC), which comprises a number of interacting scaling and leakage; see Figure 4B.
proteins, and the single strand from the dsRNA complementary In a follow-up study, Beisel et al.129 inserted RNA aptamers
to the target site. into the basal stems of shRNAs, rather than the distal loops. In
The development of RNA interference as a therapeutic tool this system, the shRNA sequence itself was inserted into the 3′
to silence gene expression has spurred the search for novel and UTR of a fluorescent reporter, allowing for direct monitoring of
improved pharmaceutical properties for medicinal purposes shRNA levels. Interestingly, the physical size of a mismatched
(reviewed by Rettig and Behlke121). The potential to introduce basal bulge was found to correlate directly with the degree of
synthetic small interfering RNAs (siRNAs) into cells and repression of the shRNA against its target, suggesting that it
tissues has led to a wide-ranging examination of siRNA provides a steric cue for processing of the hairpin. This allowed
properties, particularly their stability under hostile conditions for leakage tuning through modulation of the basal bulge size,
(such as in blood) and their silencing strength. The search has though the choice of size was limited by the requirement of
largely involved screening based on sequence and target efficient aptamer-ligand binding. Vertical scaling was achieved
sites122,123 and on chemical modifications.124−126 Efforts have by adding more copies of the shRNA in tandem onto the 3′
typically focused on identifying the strongest and most robustly UTR of the reporter gene; up to four copies were added,
silencing siRNAs, but for tuning purposes the range of separated by spacer sequences. Each added copy reduced both
characteristics across an entire library is of interest: choosing uninduced and fully induced expression levels, consistent with a
siRNAs with varying binding affinities potentially allows one to vertical scaling effect. Different spacer sequence lengths were
achieve horizontal scaling of an siRNA vs gene expression also tested, separating two copies of the shRNA in the 3′ UTR.
response curve, while choosing those with varying silencing Increasing the spacer length was found to decrease leakage
strengths has the potential to influence leakage. Thus far, a without appreciably affecting the maximal activity level in this
strong focus on finding the strongest silencers has meant that case.
other library candidates have rarely been characterized; Tunable Intergenic Regions. Pfleger et al.130 describe a
collecting data on the full range of silencing strengths obtained method for tuning the relative expression of multiple genes
from a library would provide valuable tuning information for within an operon using tunable intergenic regions (TIGRs):
future applications. intergene nucleotide sequences containing control elements
One potential method of delivering siRNAs to human cells is that include mRNA secondary structures, RNase cleavage sites,
through the bloodstream, in which case their serum stability is and RBS sequestering sequences. TIGRs are designed for
critical. Hong et al.127 demonstrated that a large proportion of placement between two genes in a polycistronic operon so that
native siRNAs are serum stable and that RNA duplexes in upon transcription, the RNase cleavage site is cut and two
serum are cleaved preferentially at two sequence-dependent distinct transcripts emerge, each containing a residual portion
dinucleotide sites, which can be avoided during design in order of the TIGR sequence (at the 3′ and 5′ ends, respectively) that
to improve stability. For siRNAs containing these dinucleotide modulates transcript stability and translational efficiency.
sites, even single modifications to the sugar backbone within Moreover, large secondary structures in the TIGR can lead to
the sites were sufficient to significantly increase stability. premature transcription termination, heavily affecting the
Varying siRNA serum stability led to horizontal scaling of the transcriptional efficiency of the second gene in the operon.
silencing response to a given dose of siRNA. By assembling and screening large libraries of TIGRs, Pfleger et
A study by Patel et al.128 compared the potency across sets of al.130 demonstrated that TIGRs could vary the relative
standard siRNA constructs used to target various sites on single expression levels of two bicistronic genes over a 100-fold
genes essential for cell growth. Although the general trend was range (offering in principle vertical scaling of an expression
that longer, chemically modified siRNAs yielded more effective response curve). Furthermore, they simultaneously tuned the
silencing, the specific target sequence also played a substantial expression of three genes within an operon encoding a
role, leading the authors to suggest that the length, chemical heterologous mevalonate biosynthetic pathway in E. coli in
modification state, and target site sequence should all be order to optimize its output flux.
considered as factors in an siRNA’s level of silencing. Carrying Protein-Based Systems. Another recent development is the
out short-term growth assays showed horizontal scaling over use of protein-based systems to implement control at the post-
an order of magnitude of siRNA concentrations. transcriptional level. Stapleton et al.131 described such a system,
Using an in vivo method for controlling siRNA concen- built around the regulatory protein L7Ae, that enabled tunable
tration, Beisel et al.117 described the use of small hairpin RNA translational repression. Provided that the recognition sequence
(shRNA)-based switches that respond to chemical induction for L7Ae (or a variant thereof) is present upstream of the
Figure 5. Tuning of enzymatic reactions. (A) Michaelis-Menten kinetics for an enzyme-catalyzed reaction: the rate of the reaction x→y varies with
the input concentration of the substrate, x, and with the enzymatic catalyst’s effective concentration and catalytic efficiency: Vmax = kcatE. (B) The
relationship between Vmax and the input ligand can be characterized (and itself tuned): each marked point, i−iv, on the Vmax vs ligand concentration
curve yields a different response curve shown in panel A. (C) A protein switch, in which the protein of interest (POI) contains autoinhibitory
domains that bind and inactivate the enzyme’s catalytic activity, reducing the enzyme’s effective concentration. The presence of a competitively
binding ligand can relieve the inhibition and restore catalytic activity.
coding region of interest, the L7Ae protein will sterically block their physical structure, and the complexity of this structure-
translation of the downstream sequences. The repression is function relationship makes rational protein design challenging.
entirely translational and does not affect the expression of other These issues mean that there are as yet fewer examples of
cistrons translated from internal ribosome entry sites (IRESs), tuning available in the post-translational space than in the
nor is it expected to appreciably modulate the degradation rate previous two levels of regulation.
of the transcript. In vivo tunability was achieved via an informed Dose-Responsive Enzymatic Catalysis. Consider a process
trial-and-error process: the wild-type L7Ae binding sequence describing an enzyme-catalyzed biochemical reaction, where the
was mutated with the intent of reducing the repressive strength process input is the amount of available substrate, and the
of the interaction to varying degrees compared to the wild-type output response is the reaction rate. Such a response curve is
interaction. The authors achieved considerable horizontal often described by well-known Michaelis-Menten kinetics
scaling, but the range of experimental results did not make it wherein the reaction rate varies from zero to some saturating
possible to determine if they also obtained vertical scaling; value Vmax = kcatE (for effective enzyme concentration E) as the
basal expression remained largely unchanged across all trials. substrate concentration increases (see Figure 5A). In our Hill-
One apparent advantage of protein-based translational control function notation, such a process has k′ = 0, k = Vmax, and n = 1.
over RNAi-based strategies is that the protein-based systems (We offer this function as an example of an enzyme-catalyzed
may require relatively less ancillary machinery thereby reducing response curve, but please note that the assumptions
the risk of saturating dynamics, whereas in the case of RNAi, it underlying the Michaelis-Menten equation will not apply to
has been observed that multiple targets or multiple shRNAs in all post-translational systems. Signaling cascades in particular
the same cellular system occasionally saturate the RNA-induced will tend to violate the assumption that the catalyzing enzyme is
silencing complex responsible for transcript regulation.132 present in much lower concentrations than a target substrate.)
Post-Translation. Biological devices that respond by Many naturally occurring enzymes change their catalytic
producing new proteins are forced to operate on long time- activity as a function of the binding of some intra- or
scales (minutes to hours or even days) by the delays inherent in extracellular ligand. This results in a change in the effective
the processes of transcription and translation. Post-translational value of Vmax, thereby vertical scaling the Michaelis-Menten
systems, involving protein-protein interactions, can respond response curve (see the relationship between Figures 5A and
much more quickly (fractions of a second to minutes) to B). (In this special case with k′ = 0, this also represents vertical
changing inputs. Unfortunately, this increased speed currently extension.) Therefore, this can be seen as a form of response
comes at the cost of significantly increased difficulty in tuning curve tuning, where the tuning strength is dependent on ligand
protein function.5,133,134 Transcriptional initiation and much of concentration.
post-transcriptional processing are highly modular and Protein Switches. Designers needing to control processes
accessible through well-established molecular biology protocols, whose enzyme catalysts do not natively respond to any ligand
allowing designers to freely substitute promoters, coding may benefit from reengineering of the enzyme itself. Protein
regions, and untranslated regions while keeping basic switches are enzymes engineered to have inducible ON and
functionality largely unchanged. Proteins, by contrast, function OFF states in terms of their catalytic activity: the binding of a
through chemical interactions that are strongly dependent on ligand flips individual proteins between their active and inactive
Figure 6. Examples of experimental tuning curves for post-translational regulation. (A) Steepness tuning by varying the number and binding affinity
of an autoinhibitory domain in protein switches.27 Reprinted by permission from Macmillan Publishers Ltd.: Nature Biotechnology, Dueber et al., 25,
660−662, copyright 2007. (B) Steepness tuning and horizontal scaling by varying the binding affinity of a decoy domain in a peptide system.30
conformations. This permits the tuning situation described tion. The conformation of the system was determined through
above, where effective V max is tuned through ligand fluorescence measurements, with the looped state defined as
concentration. In many such systems, the ligand vs Vmax inactive and the loop-free state defined as active. Decoy sites,
dose-response curve is also easily tunable. Additional coverage able to bind SH3 without affecting the activation state of the
of protein switches may be found in several recent re- system, were then added to the peptide and shown to have an
views.135−138 extensive ability to tune the relationship between the peptide’s
Most strategies for generating protein switches involve fusing activation state and the level of SH3 present. Use of a single
or inserting modular domains into the protein of interest such autoinhibitory PDZ pair resulted in a noncooperative dose-
that they disrupt or facilitate, either sterically or conformation- response to free SH3, while varying the numbers and binding
ally, the activity of the target protein. Examples of inputs for affinities of the decoy domains led to a variety of dose-
this type of regulation are small molecules,139 light,140−143 responses to SH3, implementing horizontal scaling and
ions,144 and redox conditions.145 One important approach is to steepness tuning (Figure 6B).
construct an autoinhibitory pair of domains that dimerize and Structure Rescue. Structure rescue is another promising
inhibit protein activity when no competitive ligand is present. strategy for tuning enzymatic activity. Allosteric control is
In the presence of the ligand, the dimerization is disrupted, engineered into an enzyme by structurally weakening the
allowing the protein to become active (Figure 5C); we describe enzyme through mutation to the point that its enzymatic
two such switches, below. activity is abolished, then identifying a small molecule able to
Dueber et al.27 controlled the activity of the actin bind to the mutant protein and restore its original structure.
polymerizing protein N-WASP by fusion of both an SH3 Average enzymatic activity therefore becomes tunable through
protein domain and an SH3-binding peptide, such that in the the concentration of the small molecule, providing inducible
absence of competing (nonfused) SH3-binding peptides, N- vertical scaling of the Michaelis-Menten curve via control over
WASP was autoinhibited and rendered inactive. The dose- Vmax. Deckert et al.146 successfully restored β-glycosidase
response between free SH3-binding peptides and active N- activity in a W33G mutant by rescue with indole, adopting
WASP was showed to be tunable by controlling modular the approach that the small molecule inducer should be exactly
components such as the number of SH3 domains and SH3- complementary to the residue(s) missing in the mutant protein,
binding peptides fused to each N-WASP, as well as their to achieve structure rescue. They focused on buried and tightly
binding affinities. The primary result of doing so was steepness packed tryptophan residues that, when mutated to glycine and
modulation, as shown in Figure 6A; however, steepness was supplemented with indole, yielded a protein structure highly
difficult to manipulate independent of secondary changes to similar to the original. The best mutant was able to fully restore
horizontal scaling, vertical shifting, and vertical scaling. The original activity levels in the presence of sufficient indole and
autoinhibitory paradigm was also expanded to include PDZ and thus was exogenously tunable from effectively zero activity to its
GBD interaction domains, which were subsequently assembled wild-type level.
together to regulate N-WASP under the control of a three- Protein Half-Life Modulation. We have focused mainly on
input AND gate operating on fast time-scales. rates of production of biochemical species, but of course one
Lu et al.30 reported results on a peptide system in which an can also treat degradation as its own process with a response
autoinhibitory PDZ domain was fused to a binding domain for curve (typically Michaelis-Menten) to be tuned. To this end, a
SH3 peptides. In the absence of SH3, the PDZ formed a loop number of techniques have been developed. One method
structure, while binding of SH3 to its domain prevented this involves tagging proteins with a short amino acid recognition
autoinhibition, placing the peptide into a loop-free conforma- sequence to induce degradation by an alternative set of
Figure 7. Schematics describing the general biochemical network designs reviewed in the Network Extension section. (A) A two-promoter construct
containing an interchangeable sensitivity tuner, which comprises the gene for a transcriptional activator protein and its cognate promoter. Our
schematic has been drawn based on the description provided in ref 163. (B) From ref 164. Transcriptional cascade comprising one, two, and three
repression stages. Hooshangi et al., Proc. Natl. Acad. Sci., 102, 3581−3586. Copyright 2005 National Academy of Sciences, U.S.A. (C) From ref 28.
An internal feedback loop is engineered into the yeast mating MAPK pathway via the downstream expression of a pathway modulator protein that
binds to the Ste5 scaffold protein through a leucine zipper interaction. Feedback polarity is determined by the choice of either a positive or negative
modulator, which up- or down-regulates the MAPK cascade, respectively; feedback gain is tuned by varying promoter strength or varying the leucine
zipper domains to affect binding affinity. From Bashor, et al., Science, 2008, 319, 1539. Adapted with permission from AAAS. (D) From ref 165. Two
coupled positive feedback loops are engineered into a two-component signaling system via downstream expression of both the transmembrane
receptor and intracellular TF proteins. Adapted by permission from Macmillan Publishers Ltd.: Molecular Systems Biology, Palani et al., 7, 7, copyright
2011. (E) From ref 166. Two parallel pathways constitutively repress the expression of an output protein. One pathway uses a TF repressor to
repress transcription, while the other pathway represses translation using an shRNA to induce RNAi-mediated mRNA degradation. In the bottom
schematic, a single input signal down-regulates TF and shRNA production thereby up-regulating expression of the output protein. Adapted from Cell
130, Deans et al. A tunable genetic switch based on RNAi and repressor proteins for regulating gene expression in mammalian cells, 363−372.
Copyright 2007, with permission from Elsevier.
proteolytic machinery. The ssrA tag, for example, induces peptide ligands in order to enhance the production of
degradation catalyzed by the ClpXP protease system,147 and mevalonate and glucaric acid. Varying the number of
control of some combination of the tag sequence variation, the
interaction domains fused to the scaffold allowed for
ClpXP protease level, and the level of SspB (an optional
adaptor protein) has been shown to effectively tune both Vmax optimization of the stoichiometry between the recruited
(vertical scaling) and the Michaelis constant K (horizontal enzymes, resulting in a 77-fold improvement in mevalonate
scaling).148−151 production, as well as a 3-fold improvement in glucaric acid
Tunable degradation has also been achieved by adding
production (despite already high yields). Note that in this
modular domains to proteins that promote proteolytic
degradation in the presence (or absence) of a bound ligand. study, the relationship between the scaffold concentration and
Most such ligands are small molecules, including Shield total product production was nonmonotonic, rising to a peak at
ligands,152,153 hydrophobic libraries for Halo-tags,154 auxin,155 intermediate scaffold concentrations. Although the variation of
and trimethoprim (TMP).156 the interaction domain stoichiometry resulted in prominent
Scaffolding. Proteins, DNA, and RNA have all been used to
construct “scaffolds” that co-localize multiple interacting changes to peak height and location, these response curves are
molecules by assembling them into a single physical complex, not captured by the sigmoidal response curves we have focused
enhancing interaction rates. In simple cases, this would serve to on in this review.
tune the effective Vmax of a biochemical reaction, providing DNA and RNA molecules can also be used as scaffolds. For
vertical scaling for the reaction process.
example, enzymes can be fused to zinc fingers or other
Dueber et al.157 built synthetic protein scaffolds bearing
modular SH3, PDZ, and GBD interaction domains that programmable DNA-binding domains158−160 and RNA ap-
spatially recruit three metabolic enzymes tagged with cognate tamers can be designed to bind enzymatic partners.161
ACS Synthetic Biology
■
Review
BEYOND RATE TUNING The system was then tuned by adjusting the polarity and
strength of the feedback signal: the former by using either a
Our final sections address topics that diverge from the issue of
positive or negative modulator protein and the latter by
tuning production rates but do bear on the broader problem of
changing the strength of either the leucine zipper interaction
systematically creating biological devices: the use of network
(using zipper pairs with varying Kd) or the promoter
structure effects to tune net steady-state response curves and
controlling modulator expression (both to the same effect).
the creation of consistent, modular systems.
Positive feedback was shown to vertically scale (upward) and
Network Extension. Extending or restructuring the
steepen (from n∼0.12 to 2.42) the overall network’s steady-
internal signaling network of a process can introduce complex
state response curve while negative feedback resulted in vertical
and significant transient dynamics that cannot be accurately
scaling (downward). Furthermore, by having the feedback
described by eq 1. For example, relatively simple feedforward or
positive modulator displace a constitutively expressed negative
feedback connections can give rise to delayed responses,
modulator, response curve steepness was further increased to
oscillation, or temporal adaptation;162 this implies time-
n∼2.84.
dependent rate response curves. Although important in many Feedback Loops: Bistability. Through the addition of a pair
design scenarios, such behavior is beyond the scope of this of coupled positive feedback loops, Palani and Sarkar165 were
review. In this section, we address only rate response curves for able to engineer bistability into a two-component signaling
which this behavior is insignificant, focusing our attention on system (a transmembrane receptor that signals an intracellular
how network extension has been used to tune a process’s TF via phosphorylation). The switching between stable fixed
steady-state response curve. points allowed for steady-state response curves with very high
In principle, network extension is a strategy limited only by steepness; in some cases, apparent Hill coefficients of n > 20
the availability of orthogonal parts that can be tuned into were achieved. In this network, shown in Figure 7D,
similar ranges of responsiveness; in terms of a steady-state extracellular ligand binding to the transmembrane receptor
response, the size of a process’s internal network structure is activates the intracellular TF; the activated TF then activates
irrelevant. Here, however, we review a few examples where receptor expression (the first positive feedback loop) as well as
network extension is mainly confined to the addition of a single its own expression (the second positive feedback loop).
component or network node. Experimental implementation in S. cerevisiae used the trans-
Linear Cascades: Sensitivity Tuners. A simple example of membrane receptor CRE1 from A. thaliana to bind the
network extension is provided in the work of a University of cytokinin ligand isopentenyl adenine (IP), and the yeast TF
Cambridge team, who constructed and characterized a set of 15 SKN7 as the intracellular TF. A variety of responses curves to
transcription-based “sensitivity tuners” in E. coli for the 2009 IP induction were obtained by varying the number of SKN7
iGEM competition.163 Each tuner is essentially a phage-derived operator site repeats in the corresponding promoters, thus
activating TF-promoter pair designed to be inserted, in series, effectively varying the positive feedback strength. Across five
between the input and output signals of a linear transcriptional variants of the network, steepness tuning with Hill coefficients
cascade in order to modify the shape of its rate response curve. ranging from n∼2 to 20 was observed accompanied by changes
Each tuner was characterized in an otherwise consistent in vertical extension.
construct described by x → A → y, where → denotes promoter Parallel Pathways: Reducing Leakage. In cases where
up-regulation, and x, A, and y represent chemical inducer, tighter repression of gene expression is desired, synthetic
phage activator, and reporter protein levels, respectively (see biologists have had success employing multiple repression
Figure 7A). Describing the rate response curve as in eq 3, they mechanisms in parallel. This has typically involved supplement-
found that, across the set of 15 tuners, K varied by an order of ing TF repression with a post-transcriptional mechanism such
magnitude, k′ was fairly consistent, k depended to a large extent as RNAi-mediated mRNA degradation. Deans et al.,166 for
on the tuner’s activator type (and less on the promoter choice), example, used short-hairpin RNAs (shRNAs) to induce RNAi
and n shifted between values of around 2.25 to 4 (experimental mediated degradation and controlled both shRNA and TF
resolution was too small to characterize n with much repressor production using the same input signal, as shown in
confidence), leading to horizontal scaling, vertical extension, Figure 7E. Since both the TF and the shRNA are constitutively
and steepness tuning, respectively. Another early study by produced in the absence of the input signal, the presence of the
Hooshangi et al.,164 investigating synthetic transcriptional shRNA pathway reduces steady-state expression leakage.
cascades comprising one, two, and three repression stages in Rinauldo et al.167 and Xie et al.25 employed similar techniques
E. coli (see Figure 7B), demonstrated that as cascade length using small-interfering RNA (siRNA) and microRNA
increases the overall steady-state response curve steepens (miRNA), respectively.
(increasing n). Consistency and Modularity. Sophisticated tuning of one
Feedback Loops: Engineered Scaffold Interactions. Bashor component in a biological system is of little use if the
et al.28 engineered feedback into the yeast mating MAP kinase component’s behavior is easily disrupted when its cellular
pathway, a post-translational signal transduction cascade context is changed. It is an ongoing challenge in synthetic
mediated by the Ste5 scaffold protein, which co-localizes biology to create systems that are consistent (able to display the
signaling molecules by assembling them into a single physical same behavior repeatedly, and in the face of global background
complex, thereby promoting correct pathway connectivity. An variations such as differing cell strains) and modular
internal feedback loop was generated by expressing a pathway (maintaining their behavior when linked with other engineered
modulator protein as a downstream product of the signaling systems).3,18,168−172
pathway and recruiting it back to the upstream pathway via an A variety of phenomena wherein one transcriptional process
artificial binding site on Ste5 created by fusing cognate leucine suppresses a second transcriptional process are collected under
zipper interaction (heterodimerization) modules to the scaffold the label of transcriptional interference.173 This includes
and modulator proteins (see Figure 7C). physically proximate promoters competing directly for RNAP
access, RNAPs initiating transcription from one promoter from upstream effects; by cleaving at the inserted cleavage site,
colliding with other RNAPs or blocking them from transcribing Csy4 produces processed mRNA strands that are more
from a downstream promoter, and post-transcriptional independent of their upstream genetic context and thus yield
interactions such as RNAP inactivation or RNA interference more consistent protein levels when translated.
leading one transcriptional process to reduce the effective Ribozyme-Based Insulator Parts. A study by Lou et al.178
transcriptional or translational rates of another. used sequences designed for ribozyme cleavage as a mechanism
Insulated Promoter-Gene Cassettes. Miller et al.174 created for buffering transcripts from their upstream context. The
a set of promoter-reporter cassettes in which they placed a mechanisms employed in this and the work by Qi et al.177 are
transcriptional termination sequence from E. coli upstream of conceptually similar, both based on cleaving away the 5′ UTRs
the promoter regions. This made use of the established ability to prevent their having an impact on gene expression levels.179
of such termination sequences to reduce transcription initiated Lou et al.178 constructed a logical NOT gate circuit based on
from promoters other than the target.175 Using one or two transcriptional regulation, and tested several different pro-
copies of the termination sequence, they reduced such external moters to drive expression of the first gene in the circuit. They
transcription by 94% and 97% respectively. In Davis et al.,45 observed that the NOT gate’s transfer function, in this case, the
transcriptional interference was minimized by providing mapping between this promoter’s activity and the downstream
insulated cassettes: promoters flanked by controlled sequences expression level of the circuit’s output protein, varied
both upstream and downstream of the transcriptional initiation substantially as a function of the promoter chosen. Screening
site (−105 to +55), thereby providing the promoter a a library of prospective insulator parts identified the cleavage
consistent functional neighborhood. Inserting a 24-nucleotide sequence RiboJ as the best insulator, and placing the RiboJ
sequence known to activate transcription in some promoters sequence between the circuit driving promoter and its gene
showed a wide range of resulting changes in activity in served to effectively eliminate the transfer function discrep-
promoters lacking the flanking insulating sequences, but the ancies. Transfer functions collapsed quite strikingly onto a
promoter outputs were very consistent when the insulating single curve independent of the promoter being used,
sequences were included. illustrating the insulating effect of removing inconsistent
Promoter Position Relative to ORI. As discussed in the untranslated regions from the start of the resulting mRNA
transcriptional regulation section, Block et al.32 studied the strands.
effect of promoter location relative to the genomic origin of Noise. A pervasive issue working against consistent behavior
replication (ORI) on gene expression levels, finding that in biological systems is the noise inherent in biochemical
promoters closer to the ORI expressed at higher levels. This reactions operating in regimes where fluctuations are not
suggests a mechanism for tuning response curves, but also averaged away. The topic of noise in biological systems is
provides a cautionary note in terms of consistency: if a beyond the scope of this review but has been extensively
promoter-gene pair is inserted into the genome at random, its reviewed elsewhere.180−184
behavior may vary significantly with distance from the ORI.
Consistent performance will require control of that positioning,
though Block et al.32 found that expression levels were robust to
■ DISCUSSION
Clearly there are many challenges to be addressed before tuning
two other forms of alteration: the orientation of the genes and in synthetic biology achieves the status it enjoys in traditional
the distance between genes and their TFs. Similar consid- engineering disciplines. Noise effects, while increasingly well
erations also apply to promoters on plasmid vectors: promoter understood theoretically, can be significant or design-breaking
position relative to the plasmid ORI will also cause variation in in experimental implementations. Coupling multiple systems in
effective copy number of the promoter and thus alter gene a biological context often has unexpected effects, despite design
expression levels. Transcription-replication interference was efforts to achieve true modularity. Biology inherently operates
observed by Mirkin and Mirkin176 who found that promoters in a more interconnected context than mechanical or electronic
could, if oriented such that they transcribed genes in the systems, making perfect functional compartmentalization
opposite direction to that in which DNA replication proceeded, correspondingly more challenging and raising the question of
cause significant interference with plasmid replication. Such how difficult it will be to combine multiple types of tuning to
interference would lower the plasmid copy number and reduce affect a target process. Biology also comes with substantial
gene expression levels. context-dependence, making it difficult to craft portable designs
CRISPR Editing. An alternative method of ensuring tran- that can be implemented across organisms or indeed across
scriptional consistency focuses on removing unwanted genomic variants of a single species, without significant risk of
elements from the mRNA transcript itself, after transcription their operation being disrupted by the local context.
but before translation. Qi et al.177 made use of the bacterial For all of these challenges, synthetic biology is on a
clustered regularly interspaced short palindromic repeat promising path, with the library of parts, methods, and
(CRISPR) system to protect the expression of target genes approaches growing rapidly, year by year; the already extensive
from upstream effects. Screening a randomly generated library list of tuning options available to designers in synthetic biology
of 30-nucleotide 5′ UTRs upstream of the RBS and gene seems certain to continue to grow. A combination of awareness
coding sequence of a fluorescent reporter gene, they measured of tuning applications and improved experimental methods may
gene expression of the reporter in E. coli, and observed a wide lead experimentalists to report full tuning curves more
range of expression levels across the library, suggesting variable consistently and to characterize their systems in terms of
levels of transcriptional interference induced by the UTRs. rates of change in addition to steady-state values. Our hope is
Inserting a CRISPR cleavage site targeted by the Csy4 protein that evaluating the tuning potential of a new method or system
reduced the observed relative standard deviations of reporter will come to be a standard part of the process of characterizing
expression by nearly 3-fold in the presence of the Csy4 protein, it and that libraries of components will be augmented with
suggesting that the designed system acts to insulate the mRNA collections of tuning methods, increasing the range and
sophistication of solutions available for synthetic biology design (20) Slusarczyk, A. L., Lin, A., and Weiss, R. (2012) Foundations for
problems. the design and implementation of synthetic genetic circuits. Nat. Rev.
■
Genet. 13, 406−420.
AUTHOR INFORMATION (21) Ang, J., and McMillen, D. R. (2013) Physical constraints on
biological integral control design for homeostasis and sensory
Corresponding Author adaptation. Biophys. J. 104, 505−515.
*E-mail: [email protected]. (22) Kramer, B. P., Fischer, C., and Fussenegger, M. (2004) BioLogic
Notes gates enable logical transcription control in mammalian cells.
The authors declare no competing financial interest. Biotechnol. Bioeng. 87, 478−484.
■
(23) Anderson, J. C., Voigt, C. A., and Arkin, A. P. (2007)
ACKNOWLEDGMENTS Environmental signal integration by a modular AND gate. Mol. Syst.
Biol. 3, 133.
Funding has been provided by the Natural Sciences and (24) Bagh, S., Mandal, M., and McMillen, D. R. (2010) Minimal
Engineering Research Council of Canada (NSERC) and the genetic device with multiple tunable functions. Phys. Rev. E 82, 021911.
Ontario Research Fund (ORF).
■
(25) Xie, Z., Liu, S. J., Bleris, L., and Benenson, Y. (2010) Logic
integration of mRNA signals by an RNAi-based molecular computer.
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Tuning Response Curves for Synthetic Biology

ABSTRACT: Synthetic biology may be viewed as an eﬀort to

system design in all ﬁelds of engineering, and synthetic biology

S ynthetic biology includes a concerted eﬀort to formalize an

(see Figure 1A). Nonlinear, monotonically increasing curves of dy

563 dx.doi.org/10.1021/sb4000564 | ACS Synth. Biol. 2013, 2, 547−567

564 dx.doi.org/10.1021/sb4000564 | ACS Synth. Biol. 2013, 2, 547−567

565 dx.doi.org/10.1021/sb4000564 | ACS Synth. Biol. 2013, 2, 547−567

566 dx.doi.org/10.1021/sb4000564 | ACS Synth. Biol. 2013, 2, 547−567

(171) Del Vecchio, D. (2012) Modularity in signaling systems. Phys.

567 dx.doi.org/10.1021/sb4000564 | ACS Synth. Biol. 2013, 2, 547−567

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