An Electronic Analog of Synthetic Genetic Networks

An electronic analog of a synthetic genetic network known as the repressilator is proposed. The repressilator is a synthetic biological clock consisting of a cyclic inhibitory network of three negative regulatory genes which produces oscillations in the expressed protein concentrations. Compared to previous circuit analogs of the repressilator, the circuit here takes into account more accurately the kinetics of gene expression, inhibition, and protein degradation. A good agreement between circuit measurements and numerical prediction is observed. The circuit allows for easy control of the kinetic parameters thereby aiding investigations of large varieties of potential dynamics.


Introduction
The concept of synthesizing simple gene units to realize a desired function or to reproduce a known function is new [1] in biological systems. After confirmation of the unit's desired functional behavior, a large assembly of such units can be organized to perform complex biological functions [2][3][4]. This is like engineering small integrated chips to build a computer to derive a targeted function. In efforts towards engineering biological functions, a repressilator was first demonstrated as a synthetic genetic clock expressed in Escherchia coli [5] producing oscillations in expressed protein concentrations. A mathematical model based on standard chemical kinetics was also proposed that predicted the observed oscillations.
The dynamics of coupled synthetic genetic networks (SGNs) was also investigated [6][7] theoretically to understand the generation of synchronous rhythm in an assembly of repressilators via quorum sensing type interaction. Quorum sensing [8] is a form of exchanging information that a bacterial colony uses to develop a common rhythm. This quorum sensing type of indirect coupling is set-up between the SGN cells through diffusion of auto-inducing small molecules in a common medium. When the feedback via auto-inducing agents inside a SGN cell is reinforcing [7], the coupled dynamics show a state of in-phase synchrony, whereas when the feedback is repulsive, then the coupled dynamics show various possible states [9][10][11]: in-phase and anti-phase synchrony, inhomogeneous limit cycles, inhomogeneous steady states, and homogeneous steady states. Recently, in a biological experiment [12], evidence of in-phase synchronized quorum of genetic clock units was found. However, more complex features, as chaos, antiphase, and multistability in synchronous rhythm of coupled genetic clocks are yet to be observed experimentally.
Mathematical models are always a very useful tool to predict complex behaviors of dynamical systems using numerical simulations. Experimental verification of rich multistability requires an accurate knowledge of the model parameters which is often very challenging in biological experiments. An alternative experimental approach using electronic analogs of the SGN was undertaken [13][14][15] to confirm the numerical results and to search for possible coupled dynamics. Although it is difficult to simulate the biological experiment exactly in a circuit, the advantage of an electronic SGN is accessibility of system parameters and their controllability that allows a systematic exploration of known and predictable dynamics. Earlier [13][14][15] electronic circuit analogs of SGN displayed oscillations with 120u phase shifts between the oscillating variables, qualitatively in agreement with the genetic repressilator, however, the multistability of coupled SGNs was missing since access to and control of the system parameters was lacking.
In this paper, an electronic analog of the SGN is specifically designed to derive more accurate kinetic parameters of the repressilator. The goal is to control the parameters and thereby to realize desired sets of various kinetic parameters used in the simulations of the mathematical model. As a result, the circuit shows agreement between the measurements and the numerical predictions. The building block for the electronic repressilator is a circuit model for a single negative regulatory gene. This circuit shall be useful in a variety of other SGN investigations in addition to the repressilator. Designing electronic circuits of SGN also has the purpose of reverse engineering where knowledge of the biological experiments can be utilized for new technology and applications [16].

Genetic Network Repressilator
The structure of the repressilator consists of three repressive genes connected in a loop [5], with each gene producing repressor to the subsequent gene. The genes (i = 1,2,3) each produce their own mRNA, which translate the repressor protein. Gene 19s repressor inhibits transcription of gene 29s mRNA, 29s repressor inhibits 39s mRNA, and 39s repressor inhibits 19s mRNA. Taking into account standard chemical kinetics for production, degradation and inhibition, a dynamical system model of 6 first order differential equations was used for the mRNA and the protein concentrations [5].
An electronic analog of the repressilator was also proposed earlier [14][15], where they used three voltages as the variables, thus reducing the above model to a set of three differential equations. However, these circuits did not simulate the kinetic parameters of the repressilator model. A reduced three variable model is also used here but special care is taken to retain the parameters for the chemical kinetics as in the original model [5]. The reduced genetic network (RGN) repressilator is defined as, where i = 1, 2, 3 for three genes, and the loop is closed by the condition x 0 = x 3 . The product of the i th gene is x i , and its production is inhibited by x i-1 . The parameter a accounts for the maximum transcription rate in the absence of an inhibitor, b is the decay rate of protein degradation and n is the Hill coefficient for inhibition. The Hill function is commonly used to account for sigmoidal binding kinetics. In this RGN model, there is no distinction between the mRNA and the transcribed repressor protein. We find that by this reduction, the fundamental features of the SGN model are not affected.

Description of the RGN Circuit
The building block for the RGN repressilator circuit is the circuit for a single negative regulatory gene shown in Fig. 1. The desired dynamics are given by (1), where x i corresponds to the output of the circuit and x i-1 is the input. The goal is to use a circuit which accounts for the kinetics of gene inhibition, production of repressor, and degradation of repressor. The gene's product is analogous to the charge coming from the collector of the transistor and the rate of production is the transistor current. The concentration of product x i is proportional to the voltage V i across the capacitor, and the rate of decay is the current through R C . The kinetics of gene inhibition is determined by the circuitry that couples the input voltage V i-1 (the inhibitor) to the voltage at the base of the transistor.
Circuit Analysis. Here the circuit parameters are determined that correspond to particular values of kinetic parameters a, b i , and n in (1). The dynamical equation for voltage V i is where I t (V i-1 ) is the transistor's collector current and V i-1 is the variable input voltage. The equations are expressed in dimensionless form using dimensionless time t/t where the time-scale is chosen by t = R C C 0 . The capacitor value is then where the dot denotes time derivative in dimensionless time t. In order for the circuit to model the gene kinetics it is desired that I t (V i-1 ) approximates the Hill function, where x i-1 = V i-1 /V th and V th represents an equilibrium constant for binding of repressor x i-1 to the gene's DNA. I max is the maximum current through the transistor corresponding to gene transcription in the absence of an inhibitor. The production is half-maximal, Dividing both sides of (3) by V th , the a and b i are given by a = I max R C /V th and b i = C 0 /C i . Note that I max is not due to saturation of the transistor since the voltage I max R C is chosen so that the emitter-collector voltage across the transistor does not reach zero. Instead I max is due to saturation of the op-amp as discussed below. Next we determine how the Hill coefficient n relates to the circuit parameters. The Hill function behavior consists of a transition of V i from one value to another one as V i-1 increases, with the transition occurring in the region around V i-1 = V th as shown in Fig. 2. In addition the slope in the transition region is not  constant. The circuit using the two op-amps U 1 and U 2 approximates this behavior by saturating the op-amp output and by using different gains in the transition region, a larger gain for V i-1 , V th and smaller for V i-1 . V th . Op-amp U 1 is configured as a subtraction amplifier with output G 1 (V i-1 2 V th ) = G 1 DV where G 1 is negative. Op-amp U 2 has different gains G +2 and G -2 depending on the sign of DV. Taking saturation of the outputs into account gives the voltage at the output of U 2 , where V 6sat are the saturation levels. The next step is to consider how GDV controls the transistor current. The voltage drop across R b1 is where the forward bias voltage drop across the diode is 0.6 V and V CC is the supply voltage, +5 V. The diode in series with R b1 compensates for the transistor's emitter-base voltage drop, so that the voltage across R b1 is approximately the same as the voltage across R E . The current I t (V i-1 ) through R E and the transistor is therefore (6) divided by R E . The maximum current I max occurs when the output of U 2 is saturated at GDV = V -sat , giving For comparison with the Hill function it is useful to express I t in terms of I max and the normalized input voltage x i-1 , where Dx i-1 = (x i-1 21). In order to approximate the Hill function the overall gain G 1 G -2 for V i-1 ,V th is chosen such that slope dI t /dx i-1 of the transistor current matches the slope of the Hill function at x i-1 = 1 (V i- Equating the slopes gives the condition relating Hill coefficient n to the overall gain G 1 G -2 , Choosing the gain G +2 (for V i-1 .V th ) to be less than G -2 improves the transistor current's approximation to the Hill function. We find that choosing G +2 >0.3G -2 works well for a range of parameter values a, b i , and n. Fig. 2 shows the Hill function (red dashed) and the predicted (green solid) and measured (blue dots) transistor current for n = 3.75.

Model Parameters, Circuit Parameters, and Design
Considerations. Given a circuit it is useful to be able to easily determine the corresponding model parameters. From the previous section a, b i , and n, are expressed in terms of circuit parameters by As an example, in Fig. 1 the gain for op-amp U 1 is G 1 = 26.8 and gain for op-amp U 2 is G -2 = 222 for non-saturated overall gain G 1 G -2 = 150. For V i-1 . V th the gain G +2 is approximately 26.9. For V th = 50 mV, C 0 = 1 mf, I max = 3 mA, supply V CC = 5 V, and LF412 op-amp saturation V 2sat = 23.5 V, the resulting model parameters are a = 60, b i = 1, and n = 3.8.
It is also useful to be able to determine circuit parameters that achieve a desired set of model parameters. Starting with (10), I max R C must be far enough below the supply V CC so that the emitter-collector voltage of the transistor never reaches zero. For V CC = 5 V, I max R C is chosen to be around 3 volts and the emittercollector voltage never gets less than about 1volt so that the transistor never goes into saturation. The choice of I max has some freedom. For I max = 3 mA, this determines R C = 1 kV and R E = 330 V in order to get voltage drops of 1, 1, and 3 volts across R E , the transistor, and R C , respectively, when I t = I max . The remaining free circuit parameter to adjust for a desired value of a is V th . Rearranging (10) gives For example, for I max R C = 3 V, a value of a = 100 is obtained using V th >30 mV. The capacitor value C i to use for a desired b i is easily given by (11) as The third circuit parameter is the overall gain G 1 G -2 which is determined by n and a. Rearranging (12) gives For V CC = 5 V and V 2sat = 23.5 V, then V CC -0.62 V 2sat = 7.9 V, and with I max R C = 3 V, then G 1 G -2 <2na/3. For example, if the desired parameter values are a = 100 and n = 4, then the required overall gain is G 1 G -2 <267 which can be split as desired between G 1 and G -2 . Thus, the circuit parameters that are adjusted to obtain a desired set of model parameters are V th , C i , and G 1 G -2 .
A careful selection of the op-amp is important for good approximation of the Hill function by the transistor current. The op-amp must be able to recover satisfactorily from saturation of its output. The circuit in Fig. 1 is tested with V th set to zero and with C i = 0. Results for the LF412 are shown in Fig. 3. The input voltage V i-1 (blue line) is a triangle wave from a signal generator and the measured outputs are GDV (red line) from the output of U 2 and the final output voltage V i (green line). The red curve shows that the LF412 saturates at V +sat = +4.5 V and at V -sat = 23.5 V when using a 65 V supply, and that the circuit makes the transition from high gain to low gain when V i-1 goes from negative to positive corresponding to x i-1 surpassing one. The green line shows the expected inhibitory response with respect to input V i-1 , and the maximum value of I max R C = 3 V.
Repressilator Circuit. The electronic repressilator circuit consists of three negative regulatory gene circuits (Fig. 1) connected in an inhibitory loop as shown in Fig. 4. The triangle symbol contains the 2 op-amps, transistor, and circuitry which determine parameters a and n.

Results and Discussion
The model parameters a, b i , and n are now ably determined by circuit parameters. Our results of circuit measurements and numerical predictions are shown for three cases: (1) identical genes, b-ratio = 1:1:1; (2) gene i = 1 with faster decay, b-ratio = 3:1:1; (3) gene i = 1 with slower decay, b-ratio = 0.3:1:1. Gene products are the normalized voltages x 1 (blue), x 2 (red), and x 3 (green). Circuit measurements are solid lines, numerical predictions are dashed lines. The circuit parameters V th and overall gain G 1 G -2 were varied using (13) and (15) in order to set a and n. V th varied from 30 mV to 120 mV, and G 1 G -2 from 73 to 220 corresponding to a = 25 to 100 and n = 3.0 to 6.6. The figures show results for (a, n) = (50, 6.6) and (100, 3.3). Other sets of parameters produced results with similar agreement of measurements and numerical predictions. The time constant was t = R C C 0 = (1 kV)(1 mf) = 1 ms. It follows that C i = (1 mf)/b i . Fig. 5 shows the repressilator dynamics for b-ratio = 1:1:1 where C i = 1 mf for each capacitor. The three state variables have the same shape, but with 120u phase shift. Numerical predictions are in close agreement with the measurements.
For the dynamics in Fig. 6 gene i = 1 has its capacitor reduced to 0.33 mf, so it has b 1 = 3. Thus the b -ratio for the genes in the repressilator circuit is 3:1:1. Increasing the gene product's decay rate causes larger oscillations for the product, reduced oscillations for the gene's inhibitor, and an increased oscillation frequency for the repressilator.
For the dynamics in Fig. 7 electronic gene i = 1 has its capacitor increased to 3.3 mf, so it has b 1 = 0.3. Thus the b-ratio for the genes in the repressilator circuit is 0.3:1:1. Gene i = 1 now has reduced oscillations, its inhibitor gene i = 3 has increased oscillations, and the repressilator frequency has decreased.
The circuit presented here as an electronic analog of a synthetic genetic network known as the repressilator shows good agreement between experimental measurements and numerical predictions. The circuit includes control of parameters for the Hill function which is used to model the kinetics of gene expression and inhibition in the cyclic 3-gene network. Previous electronic analogs of the repressilator [14][15] did not concentrate on the kinetics and control of the parameters, and thereby did not capture many complex dynamical features. With the ability to control the model parameters, this circuit will be useful for investigations of multistability of coupled repressilators as well as for other SGN dynamics.  Fig. 1 with V th = 0 and C i = 0. Input V i-1 (blue), op-amp output GDV (red), final output V i (green). The red curve shows saturation of the op-amp output at +4.5 and -3.5 V when using a +/-5 V supply. The change in slope when V i-1 = V th is also apparent. doi:10.1371/journal.pone.0023286.g003