Research Article

Cite This: ACS Synth. Biol. XXXX, XXX, XXX−XXX pubs.acs.org/synthbio

A Robust Molecular Network Motif for Period-Doubling Devices

Christian Cuba Samaniego and Elisa Franco*
Mechanical Engineering, University of California at Riverside, Riverside, California 92521, United States
*
S Supporting Information

ABSTRACT: Life is sustained by a variety of cyclic processes such as

cell division, muscle contraction, and neuron firing. The periodic signals
powering these processes often direct a variety of other downstream
systems, which operate at different time scales and must have the
capacity to divide or multiply the period of the master clock. Period
modulation is also an important challenge in synthetic molecular
systems, where slow and fast components may have to be coordinated
simultaneously by a single oscillator whose frequency is often difficult to
tune. Circuits that can multiply the period of a clock signal (frequency
dividers), such as binary counters and flip-flops, are commonly
encountered in electronic systems, but design principles to obtain similar devices in biological systems are still unclear. We
take inspiration from the architecture of electronic flip-flops, and we propose to build biomolecular period-doubling networks by
combining a bistable switch with negative feedback modules that preprocess the circuit inputs. We identify a network motif and
we show it can be “realized” using different biomolecular components; two of the realizations we propose rely on transcriptional
gene networks and one on nucleic acid strand displacement systems. We examine the capacity of each realization to perform
period-doubling by studying how bistability of the motif is affected by the presence of the input; for this purpose, we employ
mathematical tools from algebraic geometry that provide us with valuable insights on the input/output behavior as a function of
the realization parameters. We show that transcriptional network realizations operate correctly also in a stochastic regime when
processing oscillations from the repressilator, a canonical synthetic in vivo oscillator. Finally, we compare the performance of
different realizations in a range of realistic parameters via numerical sensitivity analysis of the period-doubling region, computed
with respect to the input period and amplitude. Our mathematical and computational analysis suggests that the motif we propose
is generally robust with respect to specific implementation details: functionally equivalent circuits can be built as long as the
species-interaction topology is respected. This indicates that experimental construction of the circuit is possible with a variety of
components within the rapidly expanding libraries available in synthetic biology.
KEYWORDS: synthetic biology, oscillations, period-doubling, bistability, network motif, frequency divider

the number of flip-flops employed determines the frequency

M olecular oscillators time many processes of life, from cell
division to differentiation.1,2 Evidence of the existence of
a master clock regulating multiple downstream processes is
found in many organisms from single cells to mammals.3 Yet,
molecular events with different time scales must operate
simultaneously to sustain life, suggesting the existence of robust
mechanisms that process and modulate the period of the
master clock without perturbing it. In synthetic biology,
network motifs for period (frequency) modulation of
oscillations could enable the coordination of multiple,
temporally distinct parallel processes.4,5 These devices would
also bypass the challenges encountered when tuning the period
of synthetic oscillators, and could expand their range of
operation.
The use of frequency dividers is widespread in analog and
digital computing devices and communication systems, which
are typically driven by a single, fast and robust clock. Design
principles for fequency dividers have been well-known for many
decades:6 the most common approach to achieve integer
frequency division is to interconnect multiple flip-flop elements.
A flip-flop circuit is in essence a bistable component that is
toggled between its two stable states by the input (Figure 1A1);
division factor. (Thus, a device performing a single frequency
division operation, or period-doubling, relies on a single flip-
flop.)
On the basis of this well-known design principle in
electronics, we ask if molecular period-doubling devices could
be built from a resettable bistable component. An affirmative
answer to this question has been recently suggested with coarse
numerical simulations of a few molecular networks built around
toggle switches, that could be reset by periodic inputs.7−10
However, a minimal network motif capable of period-doubling
has not been identified; in addition, operational trade-offs and
limitations of this class of devices have not been systematically
explored. These steps are essential to support experimental
implementations that could take advantage of the wealth of
bistable networks and molecular networking strategies available
in synthetic biology, both in vitro and in vivo.11−15
Here we describe and analyze a general molecular network
motif to achieve period-doubling of an oscillatory input signal.
Received: June 20, 2017

© XXXX American Chemical Society A DOI: 10.1021/acssynbio.7b00222

ACS Synth. Biol. XXXX, XXX, XXX−XXX
ACS Synthetic Biology
Figure 1. Network motif for period-doubling of oscillatory inputs.
(A1) Frequency division is commonly achieved in electronic circuits
using bistable switches that are toggled by the input signal. The D flip-
flop shown here includes a bistable switch built with two NAND gates,
as well as NAND-based negative feedback loops for toggling. (A2) We
propose a molecular network motif that resembles the architecture of a
flip-flop, and is the interconnection of two push modules and a bistable
subsystem. The bistable switch is forced to toggle between its stable
steady states in the presence of periodic inputs. (B) The push modules
are designed to include two intermediate species that generate a
negative feedback loop when connected to the bistable switch,
achieving an architecture similar to the flip-flop example in panel A1.
Dashed lines indicate alternative regulatory interactions within the
push modules, which would still guarantee an overall negative feedback
loop (the output wi of each push module should be high only when xi
is low and u is high). (C) Expected “state transitions” for each variable
in the network when the input is a sinusoid with period T.
The motif is defined by the interconnection of three elements:
the first is a two-node bistable switch, and the other two are
upstream negative feedback loops that preprocess the
oscillatory input (Figure 1A2). The outputs of the motif are
given by the bistable species of the molecular switch. We claim
that the motif is robust with respect to implementation, as long
as the required feedback loops are present; we support this
claim by demonstrating that three distinct realizations of the
circuit have the capacity to operate period doubling. The first
two realizations rely on Gardner and Collins’s bistable switch
and on negative feedback loops implemented with cooperative
transcriptional interactions,11,16 which are typically used to
model synthetic gene networks in vivo. The third realization is
based on a bistable switch and feedback loops built with
noncooperative interactions (uni- and bimolecular chemical
reactions),17,18 which are commonly adopted when modeling in
vitro DNA and RNA-based circuits.4,19−23
We show that, as their electronic counterpart, molecular
frequency dividers can process inputs having frequency,
amplitude, and baseline within a given range. To characterize
this “input-output rating” we combine mathematical analysis
B DOI: 10.1021/acssynbio.7b00222
Research Article
and numerical simulations. In particular, we rely on algebraic
tools to determine how the input affects bistability of the core
of the network motif.24 First, we use Sturm’s theorem to obtain
exact parametric expressions that describe the bistability region;
then, we use these exact expressions to identify how inputs can
alter the bistability region and the capacity of the circuit to
perform correctly, because the input baseline and amplitude can
be viewed as time-varying perturbations of some of the bistable
circuit parameters. In parallel, we use numerical simulations to
compute the temporal response of each network to a variety of
periodic inputs to evaluate their input-output rating. Period-
doubling of in vivo transcriptional realizations is also tested in a
stochastic regime where the input is provided by a stochastic
simulation of Elowitz’s repressilator.25 For each realization we
adopt parameters that are in a realistic range for either in vivo
artificial genetic circuits11 or in vitro nucleic acid systems.20,21
1. METHODS
1.1. Design of the Network Motif. Taking inspiration
from the structure of electronic frequency dividers, as the
circuit shown in Figure 1A1, we propose the network motif in
Figure 1A2 to build molecular frequency dividers: the core of
the architecture is a bistable switch, whose states are expected
to toggle periodically in response to a periodic stimulus. The
outputs of the network are the components of the bistable
switch that exhibit a bistable behavior (in isolation); when
toggling periodically, the outputs are expected to have opposite
phase because their stable equilibria reach either a “low” or
“high” state while never being simultaneously low or high, as
highlighted in the qualitative phase diagram in Figure 1A2
(purple dots represent stable steady states). Toggling of the
bistable switch in response to a periodic input is mediated by
circuit components we name “push modules”. The network
topology of each component (bistable switch and push
modules) is in Figure 1B. Here, pointed arrows connecting
two nodes mean that an increase in the concentration of the
species associated with the start node causes an increase in the
concentration of the species at the end node; conversely,
hammer-head arrows indicate that an increase in the
concentration of the start species causes a decrease in
concentration of the end species. Feedback loops are
highlighted in blue, and are akin to those shown in the
electronic circuit in Figure 1A1.
The outputs w 1 and w 2 of the push modules are
synchronously driven by the oscillatory input u and passed to
the bistable circuit; the push modules receive the states of the
bistable switch as inputs, generating two negative feedback
loops. We illustrate the desired toggling process of the network
elements with the support of Figure 1C, where we consider an
example oscillatory input u having period T. When the input
signal u increases during a cycle, the output wi of each push
module should increase. However, each push module is also
controlled by the switch-driven negative feedback loop: if the
ith node of the switch is in its high state, the output wi of the
push module is inhibited by the intermediate variable yi. The
presence of the intermediate species yi introduces a time lag in
the push reactions to allow toggling.9 Suppose, for instance,
that initially the bistable switch is in a state we indicate as S1,
where x1 is at its low steady state value, and x2 is at its high
steady state value. As u increases in its first cycle, w1 increases
(while w2 remains low due to the high value of x2); thus, w1
pushes x1 to become high, and forces the bistable switch to
toggle to equilibrium S2, where x1 is high and x2 is low; this
ACS Synth. Biol. XXXX, XXX, XXX−XXX
ACS Synthetic Biology Research Article

Figure 2. A transcriptional realization of the frequency divider motif that relies on competitive interactions. (A) Illustration of the gene network
corresponding to model (2)−(3); the bistable switch genes are controlled by a single promoter, for which species xi and wi compete. (B) Top:
Example integrated solutions x1 and x2 of model (2)−(3) when the input is the gray sinusoid u. Bottom: Equilibrium conditions of the bistable
switch (SI Section 2.2.2) change over time, and their number of intersection changes; variables wi are taken as time-varying parameters; at the peak
value of the input (t = 5 h and t = 15 h), the system transiently loses bistability and becomes monostable. (C) The frequency divider adapts to
changes in input period, and maintains the period-doubling property. (D) Left, 500 stochastic (Gillespie) simulations showing the circuit response to
the repressilator as input signal u; right, spectral analysis shows that the period of the input is doubled (solid line: mean, shaded regions: standard
deviation). (E) The circuit doubles the period of the input in a limited range of input amplitude and frequency, shown as the dark orange region. The
area of the period-doubling region shrinks as the baseline of the input increases, as a consequence of permanent loss of bistability.

state is maintained until u decreases at the end of the cycle. The
same process repeats at the next cycle of u, which will bring the
bistable switch back to its initial stable state S1. Overall, the
period of each toggling output of the bistable switch is 2T.
In the next sections we consider three different realizations of
this network motif, and show that each of them has the capacity
to work as a period-doubling device. Thus, we claim that this
network motif is robust with respect to its biomolecular
implementation.
1.2. Modeling and Analysis Approach. The period-
doubling network motif at Figure 1B describes the desired
dynamic interactions between components that should be
satisfied by a biomolecular implementation. Because we
consider ordinary differential equation (ODE) models, we
associate the (signed) interaction pattern to the sign pattern of
the Jacobian matrix of the model.26 The distinct realizations
considered in the next sections have a sign pattern consistent
with the topology of the motif, but the specific interactions
among species have different dynamics depending on the
implementation. In particular we consider two kinds of
dynamic interactions: cooperative, which can be modeled
using Hill functions, and stoichiometric (noncooperative).
Cooperative interactions are often used to model transcrip-
tional gene networks in cells,11,25,27 while stoichiometric
interactions are well suited to model chemical reaction
networks such as in vitro nucleic acid systems.4,21−23
A bistable switch is at the core of the network motif:
bistability is a global property of a dynamical system, and it is
defined by the coexistence of three equilibria, two stable and
one unstable (Figure 1A2). (In the following, we use the words
“equilibrium” and “steady state” as synonyms.) In general,
numerical simulations are required to evaluate the bistable
C DOI: 10.1021/acssynbio.7b00222
region of a system in parameter space. However, under
assumptions that include dissipativity and positivity, certain
systems can be classified as bistable as long as they present
three (positive) equilibria, as we show in detail in the
Supporting Information (SI) Section 1.24,28,29 Exploiting this
fact, we recast the problem of characterizing bistability regions
of our systems to the problem of studying their number of
steady states. In turn, this task can be reduced to studying the
roots of equilibrium polynomial conditions in a given interval.
The bistable subsystem of all the realizations considered here
satisfies the assumptions required to identify their bistability
regions by simply counting the number of positive equilibria.
Equilibria can be found as the zeros of polynomial expressions
that depend on the system parameters and inputs. In turn,
analytical parametric conditions for a polynomial to have exactly
three zeros in an appropriate region (positive orthant) can be
found using Sturm’s theorem (SI Section 1),24 a well-known
algebraic geometry tool. We use these conditions to identify
analytically the bistable regions of our realizations as a function
of relevant combinations of the parameters.
The bistable switch in our motif is connected to additional
components forming negative feedback loops that preprocess
the input signal u. Regardless of the chosen biomolecular
realization, the push modules have an influence on the
dynamics of the bistable core, and broadly have two effects:
the (desired) effect of toggling the stable state of the circuit,
and the (undesired) effect of driving the circuit outside of its
bistable regime. To quantify these two effects, we treat the
interconnections with the push modules as time-varying
parametric perturbations on the bistable system, and evaluate
the influence of these perturbations on the bistable region. For
this purpose, we use the analytical expressions describing the
ACS Synth. Biol. XXXX, XXX, XXX−XXX
ACS Synthetic Biology Research Article

bistability regions that were obtained using Sturm’s theorem, interactions can be realized using an AND logic gate for the
and we derive insights on the range of input period and production of wi, whose input are yi and u.16,10 (Because yi is
amplitude allowing the circuit to perform frequency division. repressed by xi, we expect that a high concentration of xi causes
In addition to the mathematical analysis outlined above, we a decrease in the concentration of yi, and therefore a decrease in
rely on numerical simulations to characterize the input-output wi.) The ODEs describing the push modules are
behavior of each realization (due to size and nonlinearity of the ρ
models). We integrate the ODEs using custom MATLAB y1̇ = − δy1 ,
scripts to obtain the temporal response of each realization to 1 + (x1/κ )m
periodic inputs. We also test the temporal response of some of ψ (u/κu)r (y1 /κ y)n
the realizations in a stochastic regime, using Gillespie’s w1̇ = · − δw1 ,
1 + (u/κu)r 1 + (y1 /κ y)n
algorithm.30 We quantify the period and amplitude of the
network output as a function of the period and amplitude of the ρ
input using MATLAB’s fft routine (Fast Fourier Transform): y2̇ = − δy2 ,
1 + (x 2/κ )m
because the system is nonlinear, the output frequency spectrum
is expected to include a range of frequencies distinct from the ψ (u/κu)r (y2 /κ y)n
w2̇ = · − δw2
input frequency. We classify the circuit as doubling the period 1 + (u/κu)r 1 + (y2 /κ y)n (3)
of the input only if the dominant component of the output
frequency spectrum is half the input frequency. In the SI we Here we assume that x1 and x2 are repressors for y1 and y2 with
examine how the period-doubling regions vary with respect to the same cooperativity coefficient m characterizing their mutual
changes in relevant parameters of the circuits such as reaction interactions within the bistable subsystem. Terms modeling the
rates and component concentrations. interconnection between the push modules and the bistable
switch are highlighted with boxes. Both u and yi are activators
2. RESULTS for wi, i = 1, 2. While there are many ways to build a genetic
2.1. A Transcriptional Network with Competitive Push AND gate,32 it is generally convenient to model this interaction
Signals. Our first realization of the period-doubling motif by taking the product of the two corresponding activator Hill
relies on the well-known transcriptional toggle switch by functions.16 If we abstract each chemical species as a binary
Gardner and Collins.11 Many natural network motifs operate variable, this product has the same sign pattern found in the
with transcriptional regulators, which have been exploited in truth table of an AND gate.33,34 Parameters κy and κu are
synthetic biology to build a multitude of artificial circuits.16,31 A apparent dissociation constants, ρ and ψ are maximal
schematic of the overall realization is in Figure 2A. expression rates, and r and n are cooperativity coefficients.
Gardner’s switch in isolation consists of two mutually If the maximal expression rate of wi is at least as large as the
repressing genes. If we neglect the dynamics of RNA maximal expression rate of xi (θ ≥ α), the Jacobian matrix
translation, the switch is modeled by two ODEs: (Section 2.3 of the SI file), is a sign definite matrix having a sign
pattern consistent with the regulatory interactions of the
α α network motif in Figure 1B.
x1̇ = − δx1 , x 2̇ = − δx 2
1 + (x 2/κ )m 1 + (x1/κ )m Figure 2B shows the outputs x1 and x2 of this realization,
(1) where the model was simulated using parameters listed in Table
1 and a sinusoidal input signal with period T = 10 h (gray trace
where x1 and x2 are protein concentrations, α is their maximal
expression rate, δ is the degradation rate, κ is the apparent
dissociation constant and m is their cooperativity coefficient for Table 1. Simulation Parameters for the Realizations Relying
repression. (For simplicity we assume that reaction rates and on Gardner’s Toggle Switch
cooperativity are the same for the dynamics of each gene, so the rate description value other studies
circuit is symmetric.) θ (μM/h) Production rate 3 0.1−100, refs 35, 36
To connect the push module outputs w1 and w2 to the toggle α (μM/h) Maximal production rate 1
switch we consider a competitive interaction, where xi and wi, i ρ = ψ (μM/h) Reactivation 2
= 1, 2 bind to the same promoter region. Competition yields a δ (/h) Degradation 1 0.4−1, ref 37
model with additive terms (highlighted with boxes below) κ (μM) Dissociation constant 0.2 10−5−1, refs 38, 39
introduced by the push signals: κy = κu (μM) 1
κw (μM) 0.1
α + θw1/κw
n=r Hill coefficient 1 1−5, refs 36, 40−42
x1̇ = − δx1 ,
1 + (x 2/κ )m + w1/κw m 2

α + θw2 /κw
x 2̇ = − δx 2
1 + (x1/κ )m + w2 /κw
Here kw is the apparent dissociation constant of wi to the gene
expressing xi, and θ is the maximal expression rate of xi induced
by wi.
The realization of the push modules is chosen based on the
following rationale: the push module output wi should be low
when u and xi are both high; in contrast, the push module
output wi should be high if u is high but xi is low. These
(2) in the figure). As the input goes through two cycles, each
variable of the bistable subsystem undergoes a single cycle. The
overall circuit is able to handle fluctuations in the period: Figure
2C shows that if the input period changes, the output period
adapts to maintain period-doubling. We note that, depending
on the input characteristics, the output waveform may
significantly differ from a perfect sinusoid: this is to be
expected due to the nonlinearity of the model. We classify the
output as correctly period-doubling as long as its principal
D DOI: 10.1021/acssynbio.7b00222
ACS Synth. Biol. XXXX, XXX, XXX−XXX
ACS Synthetic Biology
frequency component is half the input frequency (spectra are
computed using MATLAB’s fft routine).
The circuit can also operate in a stochastic regime: Figure 2D
shows an ensemble of 500 trajectories of output x1 obtained
using Gillespie’s algorithm30 to simulate the reactions
corresponding to the deterministic model (2)−(3). A stochastic
simulation of the repressilator (gray traces) was used as input to
the system25 (SI Section 5). Because in each stochastic
simulation the phase of the input and the phase of the output
may vary, the mean behavior is difficult to interpret. Phase
variations should explain the change in the mean of the
stochastic input and the variation of waveform of the mean of
the output; phase variability may also explain the increase in
output standard deviation, and the apparent similarity of input
and output periodicity. To clarify whether period doubling
occurs, we plot the frequency spectrum of input and output
signals in Figure 2D (right), which shows that on average the
dominant frequency of the output is half the dominant
frequency of the input.
2.1.1. Baseline and Amplitude of the Input Signal Reduce
the Bistability Region of the Core Toggle Switch and Reduce
the Period-Doubling Regime. In the absence of input (u = 0)
the push variables w1 and w2 converge to zero and the bistable
circuit reverts to its model in isolation. To characterize the
bistability region of the switch in isolation, we find equilibrium
conditions by setting eq 1 to zero. The positive roots of the
resulting polynomial are the admissible equilibria of the system;
the circuit is bistable as long as three equilibria are found (SI
Section 2.1). Using Sturm’s theorem (SI Section 2.2), we
identify the region in parameter space where the circuit
presents three equilibria; for m = 2 (dimerizing repressors), this
region is characterized by the following inequality:
α has a large constant bias, or baseline.
>2
δκ (4)
If this condition is satisfied, then the system admits three
equilibria; this implies bistability because the system is
dissipative and positive, and stability of its equilibria depends
exclusively on the sign of the constant term of the characteristic
polynomial (SI Section 2.1).24,28
In the presence of an input (u > 0), bistability conditions can
be derived by rewriting eq 2 as follows:
α1 α2
x1̇ = m − δx1 , x 2̇ = − δx 2
1 + (x 2/κ2) 1 + (x1/κ1)m
(5)
where
α =α
(1 + ) , θ wi
α κw
κi = κ m 1 + wj /κw , i = 1, 2,
i
1
w
+ κi
w stable equilibria, and inversely proportional to the amplitude of
j = 2, 1
With this notation, and assuming m = 2, we can derive
polynomial equilibrium conditions and use again Sturm’s
theorem to identify the parameter region in which three
equilibria are present for u(t) ≥ 0. It is possible to find tight
analytical expressions describing the region of bistability as a
function of the aggregated parameters αi/κiδ, i = 1, 2, but these
expressions can only be evaluated numerically due to their
complexity (SI Section 2.2.2). In contrast, the simple (but
conservative) inequalities below describe a region where
bistability is certainly lost:
E DOI: 10.1021/acssynbio.7b00222
Research Article
αi ⎛α⎞
=⎜ ⎟
(1 + ) < 2, θ wi
α κw
i = 1, 2,
δκi ⎝ δκ ⎠
( 1 + )(1 + )
m
wj
κw
wi
κw
j = 2, 1 (6)
These expressions are similar to expression (4) derived earlier,
but depend on the time-varying variables w1 and w2, which in
turn depend on u(t). While inequalities (6) cannot be
analytically related to the input u, they clearly highlight that a
large input may cause loss of bistability even though the system
taken in isolation is fully within its bistable region in parameter
space. This observation allows us to conclude that the circuit
will not operate correctly whenever the input presents a large,
constant baseline, which would permanently push the bistable
network outside of its bistable region. However, a transient loss
of bistability due to a large-amplitude oscillation may not have
the same detrimental effect.
2.1.2. Period-Doubling Occurs with Transient Loss of
Bistability. Numerical simulations suggest that a transient loss
of bistability does not impact the correct operation of the
circuit. In Figure 2B, bottom, we plot the equilibrium
conditions of the bistable circuit as they change over time as
a function of the integrated variables w1(t) and w2(t), taken as
time-varying parameters in the equilibrium conditions. When
the input signal u is at its lowest value, the circuit admits two
stable equilibria, however when u reaches its highest value, the
circuit is in a monostable regime; we conjecture that this
temporary transition to a monostable regime promotes faster
toggling. In contrast, the circuit performance could be
compromised by permanent loss of bistability, i.e., a violation
of conditions (6) at all times. This case would occur if the input
The observations above are validated by systematic numerical
exploration of the period-doubling region. We compute the
output period and amplitude in response to a sinusoidal input
u u t π
( ) ( )
u(t ) = uB + 2A + 2A sin 2π T − 2 , where the baseline uB,
the amplitude uA and the period T are varied. Figure 2E shows
the range of input period and amplitude in which period-
doubling is achieved, assuming the nominal parameters in
Table 1. Simulations indicate that the circuit doubles the period
of the input in a bounded region of input amplitude and period.
In this region, a small input period admits a range of amplitudes
that can also be very large; however, a large input period
correlates with a small amplitude. The qualitative inverse
relationship (large period, small amplitude) can be analytically
predicted, as shown in Figure S6: we find that the duration of a
step input (during which u is constant) required to toggle the
bistable subsystem is proportional to the distance between the
the step; this result is obtained with sensible approximations
described in SI Section 2.2.4. As suggested by our bistability
conditions, the area of the period-doubling region decreases as
the constant baseline uB of the input increases. Yet, the area
appears robust with respect to variations of most circuit
parameters as shown by our sensitivity analysis, reported in the
SI Section 2.4. Notably, we find that the period-doubling region
expands for large values of the transcription rate α, and low
values of the degradation rate δ and of the dissociation
coefficient κ.
2.2. A Transcriptional Network with Noncompetitive
Push Signals. The realization based on Gardner’s toggle
ACS Synth. Biol. XXXX, XXX, XXX−XXX
ACS Synthetic Biology Research Article

Figure 3. Realization of the frequency divider motif that relies on transcriptional noncompetitive interactions. (A) Illustration of the gene regulatory
network, where two distinct promoters control the bistable switch, eliminating competition of species xi and wi like in the previous realization. (B)
Top: Example integrated solutions x1 and x2 in model (7)−(3) when the input is the gray sinusoid; because this realization exhibits long transient
dynamics (see also panel C), we show a portion of the stationary solution (when periodic behavior is reached). Bottom: Evolution of the equilibrium
conditions of the bistable switch, where species wi are taken as time-varying parameters. (C) The circuit adapts to variations in the input period,
maintaining period-doubling. (D) Left, ensemble of 500 stochastic (Gillespie) simulations of the circuit responding to the repressilator as input
oscillatory signal; right, spectral analysis shows that the input period is doubled (solid line: mean, shaded regions: standard deviation). (E)
Deterministic period-doubling region (dark orange) as a function of amplitude and period of the input. The input baseline generally reduces the area
of the region.

switch can be modified to present noncompetitive regulatory Finally, stochastic Gillespie simulations in Figure 3D suggest
interactions between the bistable subsystem and the push that the system can operate in conditions where random
modules: fluctuations affect the input and the expression levels of the
circuit components. A stochastic simulation of the repressila-
w
θ κ1 tor25 was used as input to the system, and spectral analysis of
α w
x1̇ = − δx1 , the output x1 (Figure 3D, right) show that period doubling
x2 m
1+ ( ) (1 + )
κ
w1
κw occurs as desired.
2.2.1. The Period-Doubling Region Is Robust with Respect
to Input Amplitude, Period, and Baseline. Unlike the previous
x 2̇ =
α (θ ) w2
κw
− δx 2
realization, the bistable subsystem (7) in isolation (u = 0, wi =
x1 m 0) collapses to a system whose unique equilibrium is the origin;
1 + ( ) (1 + )
κ
w2
κw
(7) this feature may be an advantage as it implies that in the
absence of stimuli the device is fully ”off”. If u is a positive
Here all the parameters are defined as in the previous section; constant, and therefore wi > 0, the system becomes bistable. If
boxed terms highlight the influence of the push modules on the the input is a periodic signal with no baseline, the system can
bistable switch. This realization could be implemented in a become transiently bistable. The example simulation at Figure 3
synthetic circuit by using distinct promoter regions (binding B, bottom, shows how the equilibrium conditions of the
sites) for xi and wi, i = 1, 2. A scheme of the interactions among bistable subsystem vary as a function of variables wi taken as
expressed proteins and promoters is shown in Figure 3A. time-varying parameters: the subsystem is bistable only when
The model of the push subsystems is unchanged with respect u(t) becomes large.
to eq 3. The Jacobian matrix (SI Section 3.2), is a sign definite The bistability region in parameter space can be studied with
matrix having a sign pattern consistent with the regulatory
the same approach used in the previous realization. In this case,
interactions of the network motif in Figure 1B, regardless of the
parameters adopted. the multiplicative terms in model (7) (boxed terms) can be
An example numerical simulation of this realization is shown treated as time-varying factors altering the transcription rate α.
in Figure 3B, top, where we used the nominal parameters listed The positive term in each equation could therefore be rewritten
in Table 1; the system responds to the gray input sinusoid with as αi(wi)/(1 + (xi/κ)m), for i, j = 1,2, i ≠ j, where αi(wi) =
a periodic signal having twice the input period. As the previous αθ(wi/κw)/(1 + (wi/κw)). Coefficients αi(wi) vary in a bounded
realization, but with a notably longer transient, this network can interval: their value ranges between zero (when u = 0 and wi =
handle fluctuations in the input period: the output response 0) and αθ (when wi → ∞). Parametric conditions for loss of
adapts to maintain period-doubling, as shown in Figure 3C. bistability are similar to expressions (6):
F DOI: 10.1021/acssynbio.7b00222
ACS Synth. Biol. XXXX, XXX, XXX−XXX
ACS Synthetic Biology Research Article

Figure 4. Monomeric regulators can be used to implement the period-doubling motif. (A) Illustration of an example implementation network, where
two enzymes (represented by blue and red circles, species X1 and X2) are inhibited and activated by RNA species. (B) Top: Example integrated
trajectories of species x1 and x2 in model (8)−(9) when the input is the gray sinusoid. Bottom: Evolution of the equilibrium conditions of the
bistable subsystems where variables wi are taken as time-varying inputs. Bistability is transiently lost at the peaks of the input signal (t = 5 h and t = 15
h). (C) The circuit outputs adapt to fluctuations of the input period, maintaining period-doubling. (D) The area of the period-doubling region (dark
orange) decreases as the baseline of the input signal increases, as observed for the other realizations (Figure 2D and Figure 3D).

⎛ wi ⎞ depend on monomeric regulators, and all reactions occurring

⎛ α ⎞ ⎜ κw ⎟<2
⎜ ⎟θ
⎝ δκ ⎠ ⎜ 1 + wi ⎟ general class of regulators is rapidly expanding, and it includes
⎝ κw ⎠
In contrast with the previous realization, a large value of wi
reduces the region where the system is not bistable (and as a
result, it increases the region where the system is bistable).
Therefore, an input baseline is expected to promote, rather than
disrupt, the achievement of period-doubling and this realization
can potentially handle larger inputs. However, for very large
and constant input wi, the boxed terms in the bistable
subsystem converge to one, therefore in the limit case the
bistable switch becomes insulated from the input wi and the
device does not perform period-doubling. Thus, as in the
previous realization, loss of bistability must be transient for the
device to operate as desired.
Computational analysis confirms our observations: the
period-doubling region shown in Figure 3E is wider than the
corresponding region for the additive push realization (Figure
2E); in addition, the region appears less sensitive to an increase
in input baseline. These simulations suggest a design trade-off:
adopting two distinct promoters, rather than a single promoter,
to connect the bistable subsystem to the downstream modules
yields a network that operates correctly in a wider range of
input period and amplitude. Sensitivity analysis of the period-
doubling region also suggests that this realization is in general
more robust than the one relies on competitive interactions (SI
Section 3.3).
2.3. A Realization Based on Monomeric Regulators.
We now consider a realization where network interactions
G DOI: 10.1021/acssynbio.7b00222
can be modeled using first or second order kinetics. This
short regulatory RNA molecules,43,44 TALEN proteins,45 and
the CRISPR/Cas system.46,47
Here, the bistable subsystem is implemented via four
molecular components: two enzymes x1 and x2, and two
regulators x3 and x4. Enzymes can be in their active (x1, x2) or
inactive state (x̃1, x̃2), and their total concentration is constant
(xtot
i = xi + x̃i, i = 1, 2). Production of the regulators is catalyzed
by the enzymes and regulators are responsible for stoichio-
metric mutual repression and titration of the enzymes: x1
produces x3, which binds to and inactivates x2; x2 produces
x4, which binds to and inactivates x1. Reactivation of inhibited
enzymes is assumed to proceed at a linear rate, and the total
concentration of enzymes is assumed to remain constant. For
simplicity, we also assume the circuit is symmetric: production,
reactivation, and degradation pathways proceed at first order
rates driven respectively by reaction rates κ, β, and δ, while
inhibition is a second order process with rate γ. The complete
list of reactions is reported in SI Section 4.1.
Regulation in the push modules is mediated by stoichio-
metric interactions as well: species wi are produced as a
function of the input u (which is not depleted in the process),
and contribute to the reactivation of x1 and x2 at rate θ. In this
realization, the negative feedback reaction within the push
modules occurs between species yi and wi (rather than xi and yi
like in the previous architectures), via sequestration reactions
having rate ζ. The components of the push modules are
degraded at first order rate ϕ. We note that reactivation
ACS Synth. Biol. XXXX, XXX, XXX−XXX
ACS Synthetic Biology Research Article

reactions are programmed so that w1 (respectively, w2) Table 2. Nominal Simulation Parameters for the Realization
displaces x4 (x3) that is bound to x1 (x2); the complex w1·x4 Relying on Monomeric Regulators
(respectively w2·x3) is inert and considered waste. Similarly,
rate description value other studies
sequestration of wi by yi, i = 1, 2 results in waste species. Thus,
molecules x3, x4, w1, w2, y1 and y2 are produced and destroyed in κ (/s) Production rate 0.6 RNA: 10−3−1, ref 54,55
the reaction network; in contrast, xtot tot Proteins: 3 × 10−3−1, ref
1 and x2 remain constant, 56.
and the enzymes can only switch between active and inactive
β (/s) 0.2
form. Using the law of mass action, we derive the following
α = ρ (/s) 0.2
model for the interconnected system:
γ (/M/s) Inhibition 5 × 105 Nucleic acids: 104−106,
⎧ tot tot
refs 21 ,57−59
⎪ x1̇ = β(x1 − x1) − γx1x4 + θw1(x1 − x1) β (/M/s) Reactivation 3 × 104
⎪ θ (/M/s) Titration 3 × 104
⎪ tot
⎪ x 2̇ = β(x 2 − x 2) − γx 2x3 Protein/Protein: 104−
⎨ 106, refs 60, 61
+ θw2(x 2tot − x 2)
⎪ δ (/s) Degradation 3.85 × 10−4 RNA: 10−5−10−3, ref 62
⎪ x ̇ = κx − γx x − δx Proteins: 10−4−10−3, ref
⎪ 3 1 2 3 3 56
⎪ x ̇ = κx − γx x − δx xtot tot
⎩ 4 2 1 4 4 (8) 1 = x2
(nM)
Concentration 100

uA (nM) 150
⎧ y ̇ = ρx1 − ζy w − ϕy (amplitude)
⎪ 1 1 1 1
⎪ tot
⎪ w1̇ = αu − ζy1w1 − ϕw1 − θw1(x1 − x1) ⎛ κ ⎞ δ 2 + γ 2 + 2γδx tot
⎨ x tot > δ /γ , β< ⎜ ⎟
⎝ 2 ⎠ δ 2 + γ 2 − 2γδx tot
⎪ y ̇ = ρx 2 − ζy w2 − ϕy
⎪ 2 2 2

⎪ ẇ = αu − ζy w − ϕw − θw (x tot − x ) When the bistable switch is interconnected with the push

⎩ 2 2 2 2 2 2 2 (9)
The Jacobian of this system is a sign-definite matrix whose
sign pattern is largely consistent with the desired network
topology (SI Section 4.3). However, binding of monomeric
regulators result in inhibitory interactions not intended in the
network motif. For example, w1 and w2 are consumed while
binding to inactive x1 and x2: this causes unintended reduction,
or repression, of w1 and w2; these parasitic interactions can be
viewed as “retroactivity” effects.4,48
This network could be built in vitro using bacteriophage RNA
polymerases T7 and SP6 as species x1 and x2, which transcribe
mutually inhibiting aptamers x3 and x4 from synthetic templates
(these aptamer sequences are published49−51); activation
reactions could be implemented via strand displacement
reactions.17,28,51 For simplicity we neglect potential titration
reactions of species x3 and x4 by species w1 and w2, which could
be eliminated by designing hairpin domains in RNA molecules
x3 and x4.19,52,53 Note that appropriate complementarity of yi
and wi does not mean complete sequence identity of yi and
aptamers xi, and thus activity of yi as an aptamer repressor.
Aptamer function is very sensitive to sequence point mutations,
which could be used to eliminate functional competition of yi
and xi. This realization is sketched in Figure 4A.
Example numerical simulations of the model show that the
output variables x1 and x2 respond to a periodic signal by
doubling its period, as shown in Figure 4B, top. The circuit
adapts to fluctuations of the input period and maintains period-
doubling as shown in Figure 4C. The parameters used for these
simulations are at Table 2. (We do not explore the system’s
behavior in a stochastic regime, because its proposed
implementation is an in vitro reaction network operating at
high copy number.)
2.3.1. Input Amplitude and Baseline Alter the Bistable
Regime. The bistability region of circuit (8) in isolation can be
established using Sturm’s theorem,24 as done in previous
sections (SI Section 4.2). Taking xtot tot tot
1 = x2 = x , bistability
occurs when
H DOI: 10.1021/acssynbio.7b00222
modules, we can rewrite the dynamics of x1 and x2 as follows:
x1̇ = β1(x1tot − x1) − γx1x4 , x 2̇ = β2(x 2tot − x 2) − γx 2x3
(10)
where βi = β + θwi, i = 1, 2. Tractable analytical expressions
describing the bistability region cannot be easily found for
distinct βi. However, it is clear that the input-perturbed
recovery rates βi increase as a function of wi, therefore
adjustment of other parameters would be required to keep the
bistability area unchanged (relative to the system in isolation).
If all reaction rates are fixed, a constant input can cause
permanent loss of bistability; a periodic input can transiently
push the system outside of its bistable regime as highlighted in
Figure 4B where we plot the equilibrium conditions of the
bistable subsystem as a function of wi taken as time-varying
parameters (which depend on u(t)).
We numerically characterized the range of input amplitude
and period for which the circuit achieves period-doubling, as for
the other realizations. The period-doubling region drastically
decreases if the input baseline is large as shown in Figure 4D
(nominal parameters are listed in Table 2). SI Section 4.4
includes a complete sensitivity analysis; the system is
particularly sensitive to production and degradation rates of
some of the species, and cannot perform period-doubling on
any input if the production rate κ becomes large, or if the
degradation rate δ or production rate ρ are too low (all other
parameters being unchanged).
The amplitude of the output in the period-doubling region is
constant, as shown in the example simulations at Figure 4B
(top) and C. This is due to the fact that the outputs of the
bistable switch are molecular species whose total concentration
remains constant and determines the maximal distance between
the stable equilibria of the switch in isolation. This feature
makes it possible to obtain a robust output amplitude, which
can be prescribed by tuning the bistable subsystem in isolation,
and rejects perturbations in the input amplitude.
ACS Synth. Biol. XXXX, XXX, XXX−XXX
ACS Synthetic Biology Research Article

Figure 5. Performance comparison of the realizations of the period-doubling motif. (A and B) Output amplitude as a function of input amplitude
and period. (C and D) Output period as a function of input amplitude and period. We assumed zero baseline in all simulations; if input amplitude
(period) is varied, the period (amplitude) is fixed as specified on top of the panel. These comparisons highlight salient advantages and disadvantages
of each realization. The transcriptional competitive and noncompetitive realizations exhibit a tunable output amplitude, which is instead constant for
the monomeric regulators realization (it is constrained by the total amount of species x1 and x2). The transcriptional competitive realization exhibits
a narrower period-doubling range relative to the alternative networks, which could help reject noisy or excessively slow inputs.

3. DISCUSSION AND CONCLUSIONS The realizations we discussed have the potential to be

We have described a molecular network motif that operates as a
period-doubling device when receiving periodic inputs. The
motif is based on coupling a bistable switch with two upstream
push modules synchonously driven by the periodic input. Each
push module introduces a negative feedback loop, and includes
two distinct species in a sequential cascade; the requirement of
having at least two components in the push module stems from
the need of a temporal delay for proper switch toggling.9
Numerical and mathematical analysis of three different
realizations indicates that this motif is structurally well suited
for period-doubling, and that it is robust with respect to the
specific biomolecular processes chosen to implement the
regulatory pattern. This robustness is important as a growing
number of alternative molecular components becomes available
to engineer complex molecular networks.63
The realizations we considered differ in the temporal
dynamics of the interactions among molecular components.
These differences determine the range of period and amplitude
of the input signal that are successfully processed to obtain an
output signal with twice the input period. In Figure 5 we
provide a performance comparison of the three realizations
when the nominal input signal is a sinusoid with no baseline (0
nM), amplitude of 300 nM and period of 15 h (realizations
were simulated using the parameters at Tables 1 and 2). The
amplitude of the outptut signal of both realizations based on
Gardner’s switch is sensitive to changes in both amplitude and
period of the input (Figure 5A and B); in contrast, the output
amplitude of the realization based on monomeric regulators is
robust to input period and amplitude, due to the fact that the
total concentration of components of the bistable switch is
constant. The period-doubling capacity of the competitive
Gardner realization is restricted to a selective range of input
period and amplitude (Figure 5C and D), while the
noncompetitive Gardner switch and the monomeric network
achieve period-doubling in a wider input range. In all cases, the
circuits appear to “filter” low-amplitude inputs (by exhibiting a
low-amplitude output): this could be a useful feature in cases
where small, noisy oscillations should not be transmitted to
downstream systems. It is likely that other realizations having
the same interconnection topology could operate correctly and
present distinct advantages depending on the molecular
context.
I DOI: 10.1021/acssynbio.7b00222
experimentally implemented. Many tunable implementations of
bistable switches exist in vivo11,12,14,45,64,65 and in vitro:15,21,66
interconnecting these devices with negative feedback push
modules is feasible as long molecular signals are transmitted in
a modular manner, avoiding retroactivity effects.4,48 A memory
element was successfully connected to a NOR gate and used to
demonstrate push-on push-off sequential logic function in
E. coli,67 where UV irradiation was processed as an external
input to toggle the bistable switch. A motif very close to our
architecture was proposed in66 to build in vitro a push-on push-
off memory device using the polymerase-exonuclease-nickase
(PEN) toolbox; the possible use of the same motif as a
frequency divider was suggested with simulations without in-
depth analysis.7
Alternative motifs for frequency division have been examined
from a computational standpoint. An approach based on
sequential connection of transcriptional logic modules was
suggested to build a genetic clock and JK flip-flop units for
frequency division; this approach requires that the circuit
parameters enable pulse width modulation of the clock signal;10
although this circuit could be built with only six orthogonal
genes, little guidance is offered in terms of implementation
feasibility. Frequency multiplication in response to periodic
signals was computationally demonstrated in a multifunctional
network;8 however, this system is based on coupling multiple
bistable systems and its implementation would require the use
of several orthogonal repressors.
The size and complexity of synthetic molecular networks is
rapidly increasing, introducing a need for devices with the
capacity to coordinate temporally the function of multiple
circuits having distinct time scales. Modular period-doubling
elements would enable the construction of large, scalable
networks controlled by a single robust clock, mimicking the
architecture of engineered computing devices.4 These devices
would also address the problem of limited tunability of many
existing synthetic clocks, and could be combined with
amplitude-processing devices68 to fully control the waveform
of periodic signals. There is also evidence that living cells
coordinate temporal patterns of gene expression globally using
one (or few) master oscillators:69−71 we conjecture that
network motifs that process and modulate the period of the
master clock signal could be crucial for correct timing of
ACS Synth. Biol. XXXX, XXX, XXX−XXX
ACS Synthetic Biology
growth, development, and differentiation. In this context, our
work suggests that bistable switches coupled to negative
feedback loops may yield robust motifs to control timing of
cellular events.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acssynbio.7b00222.
Detailed information on the mathematical methods and
the numerical simulations (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: efranco@engr.ucr.edu.
ORCID
Elisa Franco: 0000-0003-1103-2668
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors thank Dan Siegal, Diego Oyarzún, Amy O’Brien,
Elisenda Feliu, and Franco Blanchini for discussions and useful
comments. This work has been supported by the National
Science Foundation through grant CMMI-1266402.
■
REFERENCES
(1) Matsuo, T., Yamaguchi, S., Mitsui, S., Emi, A., Shimoda, F., and
Okamura, H. (2003) Control mechanism of the circadian clock for
timing of cell division in vivo. Science 302, 255−259.
(2) Pourquié, O. (2003) The segmentation clock: converting
embryonic time into spatial pattern. Science 301, 328−330.
(3) Bell-Pedersen, D., Cassone, V. M., Earnest, D. J., Golden, S. S.,
Hardin, P. E., Thomas, T. L., and Zoran, M. J. (2005) Circadian
rhythms from multiple oscillators: lessons from diverse organisms.
Nat. Rev. Genet. 6, 544−556.
(4) Franco, E., Friedrichs, E., Kim, J., Jungmann, R., Murray, R.,
Winfree, E., and Simmel, F. C. (2011) Timing molecular motion and
production with a synthetic transcriptional clock. Proc. Natl. Acad. Sci.
U. S. A. 108, E784−E793.
(5) Purnick, P. E., and Weiss, R. (2009) The second wave of
synthetic biology: from modules to systems. Nat. Rev. Mol. Cell Biol.
10, 410−422.
(6) Katz, R. H., and Borriello, G. (2005) Contemporary Logic Design,
Pearson Prentice Hall.
(7) Aubert, N., Mosca, C., Fujii, T., Hagiya, M., and Rondelez, Y.
(2014) Computer-assisted design for scaling up systems based on
DNA reaction networks. J. R. Soc., Interface 11, 20131167.
(8) Purcell, O., di Bernardo, M., Grierson, C. S., and Savery, N. J.
(2011) A multi-functional synthetic gene network: a frequency
multiplier, oscillator and switch. PLoS One 6, e16140.
(9) Hillenbrand, P., Fritz, G., and Gerland, U. (2013) Biological
signal processing with a genetic toggle switch. PLoS One 8, e68345.
(10) Chuang, C.-H., and Lin, C.-L. (2014) Synthesizing genetic
sequential logic circuit with clock pulse generator. BMC Syst. Biol. 8,
63.
(11) Gardner, T. S., Cantor, C. R., and Collins, J. J. (2000)
Construction of a genetic toggle switch in Escherichia coli. Nature 403,
339−342.
(12) Chen, D., and Arkin, A. P. (2012) Sequestration-based
bistability enables tuning of the switching boundaries and design of
a latch. Mol. Syst. Biol. 8, 620.
(13) Padirac, A., Fujii, T., and Rondelez, Y. (2012) Bottom-up
construction of in vitro switchable memories. Proc. Natl. Acad. Sci. U. S.
A. 109, E3212−E3220.
J DOI: 10.1021/acssynbio.7b00222
Research Article
(14) Huang, D., Holtz, W. J., and Maharbiz, M. M. (2012) A genetic
bistable switch utilizing nonlinear protein degradation. J. Biol. Eng. 6, 9.
(15) Subsoontorn, P., Kim, J., and Winfree, E. (2012) Ensemble
Bayesian analysis of bistability in a synthetic transcriptional switch.
ACS Synth. Biol. 1, 299−316.
(16) Alon, U. (2006) An Introduction to Systems Biology: Design
Principles of Biological Circuits, CRC Press.
(17) Mardanlou, V., Samaniego, C. C., and Franco, E. (2015) A
bistable biomolecular network based on monomeric inhibition
reactions. 2015 54th IEEE Conference on Decision and Control
(CDC), pp 3858−3863.
(18) Samaniego, C. C., and Franco, E. (2015) A minimal
biomolecular frequency divider. 2015 54th IEEE Conference on Decision
and Control (CDC), pp 1277−1282.
(19) Dirks, R. M., and Pierce, N. A. (2004) Triggered amplification
by hybridization chain reaction. Proc. Natl. Acad. Sci. U. S. A. 101,
15275−15278.
(20) Seelig, G., Soloveichik, D., Zhang, D. Y., and Winfree, E. (2006)
Enzyme-free nucleic acid logic circuits. Science 314, 1585−1588.
(21) Kim, J., White, K. S., and Winfree, E. (2006) Construction of an
in vitro bistable circuit from synthetic transcriptional switches. Mol.
Syst. Biol., DOI: 10.1038/msb4100099.
(22) Zhang, D. Y., and Seelig, G. (2011) Dynamic DNA
nanotechnology using strand-displacement reactions. Nat. Chem. 3,
103−113.
(23) Montagne, K., Plasson, R., Sakai, Y., Fujii, T., and Rondelez, Y.
(2011) Programming an in vitro DNA oscillator using a molecular
networking strategy. Mol. Syst. Biol. 7, 466.
(24) Siegal-Gaskins, D., Franco, E., Zhou, T., and Murray, R. M.
(2015) An analytical approach to bistable biological circuit
discrimination using real algebraic geometry. J. R. Soc., Interface 12,
20150288.
(25) Elowitz, M. B., and Leibler, S. (2000) A synthetic oscillatory
network of transcriptional regulators. Nature 403, 335−338.
(26) Blanchini, F., Franco, E., and Giordano, G. (2014) A Structural
Classification of Candidate Oscillatory and Multistationary Biochem-
ical Systems. Bull. Math. Biol. 76, 2542−2569.
(27) De Jong, H. (2002) Modeling and simulation of genetic
regulatory systems: a literature review. J. Comput. Biol. 9, 67−103.
(28) Mardanlou, V., Tran, C. H., and Franco, E. (2014) Design of a
molecular bistable system with RNA-mediated regulation. 2014 IEEE
53rd Annual Conference on Decision and Control (CDC), pp 4605−
4610.
(29) Conradi, C., Feliu, E., Mincheva, M., and Wiuf, C. (2016)
Identifying parameter regions for multistationarity. PLoS Comput. Biol.
13, e1005751.
(30) Gillespie, D. T. (1977) Exact stochastic simulation of coupled
chemical reactions. J. Phys. Chem. 81, 2340−2361.
(31) Way, J. C., Collins, J. J., Keasling, J. D., and Silver, P. A. (2014)
Integrating biological redesign: where synthetic biology came from and
where it needs to go. Cell 157, 151−161.
(32) Sayut, D. J., Niu, Y., and Sun, L. (2009) Construction and
enhancement of a minimal genetic and logic gate. Applied and
environmental microbiology 75, 637−642.
(33) Moon, T. S., Lou, C., Tamsir, A., Stanton, B. C., and Voigt, C. A.
(2012) Genetic programs constructed from layered logic gates in
single cells. Nature 491, 249.
(34) Wang, B., Kitney, R. I., Joly, N., and Buck, M. (2011)
Engineering modular and orthogonal genetic logic gates for robust
digital-like synthetic biology. Nat. Commun. 2, 508.
(35) Qian, Y., Huang, H.-H., Jiménez, J., and Del Vecchio, D. (2016)
Resource Competition Shapes the Response of Genetic Circuits. ACS
Synth. Biol. 6, 1263.
(36) Basu, S., Gerchman, Y., Collins, C. H., Arnold, F. H., and Weiss,
R. (2005) A synthetic multicellular system for programmed pattern
formation. Nature 434, 1130−1134.
(37) Andersen, J. B., Sternberg, C., Poulsen, L. K., Bjørn, S. P.,
Givskov, M., and Molin, S. (1998) New unstable variants of green
ACS Synth. Biol. XXXX, XXX, XXX−XXX
ACS Synthetic Biology Research Article

fluorescent protein for studies of transient gene expression in bacteria. (58) Zhang, D. Y., and Winfree, E. (2009) Control of DNA strand
Appl. Environ. Microbiol. 64, 2240−2246. displacement kinetics using toehold exchange. J. Am. Chem. Soc. 131,
(38) Liu, M., Gupte, G., Roy, S., Bandwar, R. P., Patel, S. S., and 17303−17314.
Garges, S. (2003) Kinetics of Transcription Initiation at lacP1. (59) Zhang, J. X., Fang, J. Z., Duan, W., Wu, L., Zhang, A., Dalchau,
Multiple roles of cyclic amp receptor protein. J. Biol. Chem. 278, N., Yordanov, B., Petersen, R., Phillips, A., and Zhang, D. (2017)
39755−39761. Predicting DNA Hybridization Kinetics from Sequence. Nat. Chem.,
(39) Milo, R., Jorgensen, P., Moran, U., Weber, G., and Springer, M. DOI: 10.1038/nchem.2877.
(2010) BioNumbers − The database of key numbers in molecular and (60) Schlosshauer, M., and Baker, D. (2004) Realistic protein−
cell biology. Nucleic Acids Res. 38, D750−D753. protein association rates from a simple diffusional model neglecting
(40) Garcia-Ojalvo, J., Elowitz, M. B., and Strogatz, S. H. (2004) long-range interactions, free energy barriers, and landscape ruggedness.
Modeling a synthetic multicellular clock: repressilators coupled by Protein Sci. 13, 1660−1669.
quorum sensing. Proc. Natl. Acad. Sci. U. S. A. 101, 10955−10960. (61) Schreiber, G., Haran, G., and Zhou, H.-X. (2009) Fundamental
aspects of protein- protein association kinetics. Chem. Rev. 109, 839−
(41) Weber, W., Rimann, M., Spielmann, M., Keller, B., Daoud-El
860.
Baba, M., Aubel, D., Weber, C. C., and Fussenegger, M. (2004) Gas-
(62) Kim, J., Khetarpal, I., Sen, S., and Murray, R. M. (2014)
inducible transgene expression in mammalian cells and mice. Nat. Synthetic circuit for exact adaptation and fold-change detection.
Biotechnol. 22, 1440−1444. Nucleic Acids Res. 42, 6078.
(42) Koutinas, M., Lam, M.-C., Kiparissides, A., Silva-Rocha, R., (63) Cheng, A. A., and Lu, T. K. (2012) Synthetic biology: an
Godinho, M., Livingston, A. G., Pistikopoulos, E. N., De Lorenzo, V., emerging engineering discipline. Annu. Rev. Biomed. Eng. 14, 155−178.
Dos Santos, V. A., and Mantalaris, A. (2010) The regulatory logic of (64) Atkinson, M. R., Savageau, M. A., Myers, J. T., and Ninfa, A. J.
m-xylene biodegradation by Pseudomonas putida mt-2 exposed by (2003) Development of Genetic Circuitry Exhibiting Toggle Switch or
dynamic modelling of the principal node Ps/Pr of the TOL plasmid. Oscillatory Behavior in Escherichia coli. Cell 113, 597−607.
Environ. Microbiol. 12, 1705−1718. (65) Ajo-Franklin, C. M., Drubin, D. A., Eskin, J. A., Gee, E. P.,
(43) Waters, L. S., and Storz, G. (2009) Regulatory RNAs in bacteria. Landgraf, D., Phillips, I., and Silver, P. A. (2007) Rational design of
Cell 136, 615−628. memory in eukaryotic cells. Genes Dev. 21, 2271−2276.
(44) Schmiedel, J. M., Axmann, I. M., and Legewie, S. (2012) Multi- (66) Padirac, A., Fujii, T., and Rondelez, Y. (2012) Bottom-up
target regulation by small RNAs synchronizes gene expression construction of in vitro switchable memories. Proc. Natl. Acad. Sci. U. S.
thresholds and may enhance ultrasensitive behavior. PLoS One 7, A. 109, E3212−E3220.
e42296. (67) Lou, C., et al. (2010) Synthesizing a novel genetic sequential
(45) Lebar, T., et al. (2014) A bistable genetic switch based on logic circuit: a push-on push-off switch. Mol. Syst. Biol. 6, 350.
designable DNA-binding domains. Nat. Commun. 5, 5007. (68) Agrawal, D. K., Franco, E., and Schulman, R. (2015) A self-
(46) Nielsen, A. A., and Voigt, C. A. (2014) Multi-input CRISPR/ regulating biomolecular comparator for processing oscillatory signals. J.
Cas genetic circuits that interface host regulatory networks. Mol. Syst. R. Soc., Interface 12, 20150586.
Biol. 10, 11. (69) Klevecz, R. R., Bolen, J., Forrest, G., and Murray, D. B. (2004) A
(47) Bikard, D., Jiang, W., Samai, P., Hochschild, A., Zhang, F., and genomewide oscillation in transcription gates DNA replication and cell
Marraffini, L. A. (2013) Programmable repression and activation of cycle. Proc. Natl. Acad. Sci. U. S. A. 101, 1200−1205.
bacterial gene expression using an engineered CRISPR-Cas system. (70) Orlando, D. A., Lin, C. Y., Bernard, A., Wang, J. Y., Socolar, J. E.,
Iversen, E. S., Hartemink, A. J., and Haase, S. B. (2008) Global control
Nucleic Acids Res. 41, 7429−7437.
of cell-cycle transcription by coupled CDK and network oscillators.
(48) Del Vecchio, D., Ninfa, A., and Sontag, E. (2008) Modular Cell
Nature 453, 944−947.
Biology: Retroactivity and Insulation. Mol. Syst. Biol. 4, 161.
(71) Rahi, S. J., Pecani, K., Ondracka, A., Oikonomou, C., and Cross,
(49) Ohuchi, S., Mori, Y., and Nakamura, Y. (2012) Evolution of an
F. R. (2016) The CDK-APC/C Oscillator Predominantly Entrains
inhibitory RNA aptamer against T7 RNA polymerase. FEBS Open Bio Periodic Cell-Cycle Transcription. Cell 165, 475−487.
2, 203.
(50) Mori, Y., Nakamura, Y., and Ohuchi, S. (2012) Inhibitory RNA
aptamer against SP6 RNA polymerase. Biochem. Biophys. Res. Commun.
420, 440−443.
(51) Lloyd, J., Tran, C. H., Wadhwani, K., Cuba Samaniego, C.,
Subramanian, H. K., and Franco, E. (2017) Dynamic control of
aptamer-ligand activity using strand displacement reactions. ACS
Synth. Biol., DOI: 10.1021/acssynbio.7b00277.
(52) Green, S. J., Lubrich, D., and Turberfield, A. J. (2006) DNA
hairpins: fuel for autonomous DNA devices. Biophys. J. 91, 2966−
2975.
(53) Schreck, J. S., Ouldridge, T. E., Romano, F., Šulc, P., Shaw, L. P.,
Louis, A. A., and Doye, J. P. (2015) DNA hairpins destabilize duplexes
primarily by promoting melting rather than by inhibiting hybridization.
Nucleic Acids Res. 43, 6181−6190.
(54) Vogel, U., and Jensen, K. F. (1994) The RNA chain elongation
rate in Escherichia coli depends on the growth rate. J. Bacteriol. 176,
2807−2813.
(55) Chen, H., Shiroguchi, K., Ge, H., and Xie, X. S. (2015) Genome-
wide study of mRNA degradation and transcript elongation in
Escherichia coli. Mol. Syst. Biol. 11, 781.
(56) Buchler, N. E., and Louis, M. (2008) Molecular Titration and
Ultrasensitivity in Regulatory Networks. J. Mol. Biol. 384, 1106−1119.
(57) Zhang, D. Y., Turberfield, A. J., Yurke, B., and Winfree, E.
(2007) Engineering Entropy-Driven Reactions and Networks
Catalyzed by DNA. Science 318, 1121−1125.

K DOI: 10.1021/acssynbio.7b00222
ACS Synth. Biol. XXXX, XXX, XXX−XXX