• Introduction
• Defining The Best Topology
• Sensitivity analysis

Model

Introduction

A regular reporter (for example, fluorescent proteins or pigment) can only detect a narrow range of concentration of input signals, (Figure 1) because they mainly possess a Hill-function type dose-response curve. The linear proportion of such a dose-response curve is relatively narrow，causing it sensitive to only a narrow range of input intensity. Thus the regular reporters are not proper devices for quantitative measurement. In order to design a quantitative, economical and convenient aromatics detector, we decided to build a band pass filter.

Introduction

Here we focus on developing advanced equipment for our toolkit to implement fast, economical and convenient detection of aromatic compounds in environment.

Common reporting systems failed to meet this requirement because they often possess a Hill-function type dose-response curve. (A Hill-Function is written as

where denotes the output intensity, denotes the maximum output intensity, denotes the input intensity, equals to the input intensity corresponding to half-maximum output intensity and denotes the hill coefficient.) As can be observed from the graph of a typical Hill function (Fig.1, a), the linear range of a Hill function could be rather narrow, and the transition from low-output to high-output may be quite obscure to naked eyes. Thus appropriate equipment would be required to accurately measure output that follows Hill function type dose-response curve, making the measurement expensive and time consuming.

Figure 1. General reporting systems and the band-pass filter. (a)The output of a general reporting systems, it's hard to tell the concentration when it’s bigger or smaller than some certain concentration. (b)The band-pass filter only responses to a certain concentration.

Although unaided eyes can barely determine the absolute intensity of a signal among a series of gradually increasing ones (Fig. 1, a), humans are pretty competent at determining the strongest one among a series of signals with different intensities, especially when there is a single peak among the signals (Fig.1 b). Fortunately, a band-pass filter is exactly the equipment that can turn a series of gradually increasing input signals into a series of output signals with a single peak.

It can be expected that when a band-pass filter is successful constructed, we may serially dilute our sample into a concentration gradient and put our biosensor into the sample. The analyte concentration can be easily determined by the serial number of the test tube exhibiting highest output intensity (Fig. 2).

Thus we reasoned that by implementing a band-pass filter circuit in our bacterial host cells, we might realize fast, economical and convenient detection of aromatic compounds in environment.

Figure 2.Test using band-pass filter.

The Concept of Band-Pass Filter

Band-pass filter is a term used in electronic engineering. It describes a device that passes signals with frequencies confined to a certain range and blocks signals with frequencies outside that range. The band-pass filter is constructed by combining a high-pass filter, which only pass signals with high frequencies, and a low-pass filter, which only pass signals with low frequencies (Fig.3).

Figure 3.The sketch diagram of band-pass filter in electronic engineering. The vertical arrow shows the input and output of a high-pass filter and a low high-pass filter. The horizontal arrow shows the input and output of a band-pass filter. As shown in the picture, the input signal is processed by the high-pass filter, then the signals is passed to the low-pass filter.

In analogy to an electric band-pass filter, a biological band-pass filter is a device that can be activated only by an input signal with medium intensity. Neither signal with low nor high intensity will generate an output signal (Fig.4).

Quite similar to an electric band-pass filter, a biological band-pass filter can also be separated into two components, namely the two types of regulation the input node exerts on the output node in the network topology. In one way, the input node activates the output node through a positive feed-forward loop; in another way, the input node inhibits the output node through a negative feed-forward loop. Such a network topology, with two counteracting regulating feed-forward loop connecting input node and output node, is called an incoherent feed-forward loop topology (Fig.4 a). The positive feed-forward loop will respond only to high intensity input signal (Fig .4 b), and the negative feed-forward loop will respond only to the low intensity input signal (Fig.4 c). By fine-tuning transition points of the dose-response curve of the incoherent feed-forward loops so that the transition point of the negative loop is higher than that of the positive loop, the biological band-pass filter, constructed by combining these two loops together, will respond only to a medium intensity input signal and generate an output peak at a specific concentration (Fig.4 d).

Figure 4. The sketch diagram of a band-pass filter in biology. Node A is a transcriptional factor that expression constitutively. Node B is a transcriptional factor controlled by Node A. Node C is the reporter (like, sfGFP, RFP etc.). The input is the ligand that can bind to the transcriptional factor, the output is the fluorescence intensity.(a) Incoherent feed-forward loop that can generate a band-pass filter. The inputs are aromatic compounds and the output is the fluorescence intensity. (b)The input-output curve of the positive loop. (c)The input-output curve of the negative loop. (d)The input-output curve of the incoherent feed-forward loop.

Constructing Band-Pass Filter

To rationally design the circuit for our band-pass filter, we first selected three potential circuit networks containing a core topology: incoherent feed-forward loop.

We then used Ordinary Differential Equations (ODEs) to analyze these circuit networks to identify the most robust circuit network and finally chose to follow a four-node network.

To select appropriate proteins to function as individual nodes in the circuit network, we first figured out the most crucial parameters through a parameter sensitivity analysis and determined the most desirable value for these parameters. Then we chose regulatory proteins whose kinetic parameter values are close to the desirable values, reasoning that they would work much more efficiently than casually chosen ones.

Based on the analysis above, we selected phage transcription activator phiR73delta as internode activator and phage transcription inhibitor cI as internode inhibitor while choosing NahR as the input sensor and sfGFP as reporter. The final construct is shown in Fig Details of constructing process can be view at Model.

Construction of our hybrid promoter

After deciding the circuit and protein to use, we still need to address an important issue: we need to find a way to enable synergic regulation of the reporter gene by two different transcription regulators. So we modified bacteriophage φR73’s promoter into a hybrid promoter that can be activated by the PhiR73 delta activator and repressed by the repressor cI simultaneously and put reporter sfGFP under its regulation. We replaced the sequence between the -10 and -35 elements of the promoter with the cI binding site Or1 from Phage λ. When PhiR73δ activator binds to its binding site upstream of -35 element, the transcription will start. The binding of cI dimers will block the binding of σ70 factors and thus repress the transcription (Fig.6).

Figure 5.The Construction of Our Hybrid Promoter.

As the input signal increase, the promoter will function in the following way: when the input signal is weak, the concentration of PhiR73 delta is too low to generate a strong output; when the input signal is medium, despite a portion of promoters occupied by cI dimmers, the rest still can be activated by PhiR73 delta and bring about a visible output; when the input signal is strong, almost all of the promoters are blocked by cI dimers and the output is shut down. Hence only medium input signal can generate a significant output and the Band Pass Filter was made.