We have equipped our toolkit with a collection of biosensors, each capable of sensing a specific group of aromatic compounds (Fig. 1). Considering the complexity of practical analysis, we may need to analyze a multi-component sample using more than one biosensors. For example, a sample maybe consists of both 4-CISaA (an inducer of NahR) and 3-CIBzO (an inducer of XylS). If we want to quantify the concentrations of both 4-CISaA and 3-CIBzO, the biosensors NahR and XylS need to be applied to the sample separately. To make sure that the multi-component analysis is valid, we must guarantee that the presence of 3-CIBzO won’t interfere with the dose response of NahR biosensor to 4-CISaA, and vice versa.

If the presence of an inducer of XylS (not an inducer of NahR) doesn’t interfere with the dose response of NahR to any of its inducers, and vice versa, we call the biosensor NahR and XylS are "orthogonal"; namely, no synergistic/antagonistic effects happen between the inducers of NahR and XylS biosensors.

We examined the orthogonality between 4 representative biosensors (Fig.2). The orthogonality test between two biosensors, biosensor I and biosensor II, was performed in the following procedure:

1. A typical inducer A for biosensor I and a typical inducer B for biosensor II were selected.
2. The dose response of biosensor I to inducer A was measured, under the perturbation of inducer B.
3. The dose-response of biosensor II to inducer B was measured, under the perturbation of inducer A.

If biosensor I and biosensor II are orthogonal, the dose response of biosensor I to inducer A should be constant, regardless of the concentrations of inducer B; and the dose response of biosensor II to inducer B should be constant, regardless of the concentrations of inducer A. Namely, for two "orthogonal" biosensors, the perturbation of an unrelated inducer has negligible effect on the dose response of a biosensor to its related inducer (Fig. 3).

The orithogonality of XylS, NahR, HbpR and DmpR biosensors have been carefully confirmed using the test assay introduced above (Fig.4). The experimental points were processed by linear fitting and the slopes of the fitting curves were compared with 1. The closer the slope was to 1, the more orthogonal the biosensors were. The results showed that biosensor XylS and NahR (Fig.4a,b), XylS and HbpR (Fig.4c,d), NahR and HbpR (Fig.4e,f), XylS and DmpR (Fig.4g,h), NahR and DmpR (Fig.4i,j), and HbpR and DmpR (Fig.4k,l) are all investigated to be orthogonal, which is summarized in Fig.5.

In conclusion, we have confirmed the orthogonality among inducers of different biosensors, which is one of the main features we expect for our aromatics-sensing toolkit. Our sensors are well suited to multicomponent analysis.

Figure 1. The aromatics spectrum showing the aromatics-sensing profiles of our individual biosensors. Each color segment in the central spectrum represents the detection profile of a biosensor. Structural formula highlighted in color stand for the aromatic compounds that can be detected by our biosensors.

Figure 2. Orthogonality test assay for biosensor I and biosensor II. (a) Biosensor I was added into the test assay. Different mixtures of inducers were added into lane 1, 2, and 3 respectively as listed above. Effect of inducer B (detected by biosensor II) upon the dose-response curve of inducer A (detected by biosensor I) was tested by comparing the fluorescence intensity of biosensor I among lane 1 ,2, and 3. (b) Biosensor II was added into the test assay. Different mixtures of inducers were added into lane 1, 2, and 3 respectively as listed above. Effect of inducer A upon the dose-response curve of inducer B was tested by comparing the fluorescence intensity of biosensor II among lane 1 ,2, and 3.

Figure 3.Correlation of the inducer B and the dose-response of biosensor I to its inducer A. Each point on the right plot represents a concentration of inducer A. It's x coordinate represents the fluorescence when inducer B is 0 and the y coordinate represents the fluorescence when the cell is exposed to a none-zero concentration of inducer B. If the dose-response of biosensor I is invariant to the concentration of inducer B, the x coordinate of a experimental point should be equal to its y coordinate and the experimental points are supposed to be aligned in a line whose slope is one.

Figure 4. Experimental points and the linear fitting curves of the orthogonality test. The black dashed lines are with the slopes of 1, showing as the reference line. The slopes of the experimental fitting curves were showed in the upside portion of the figure, all of them were around 1. These data showed the orthogonality among biosensors(a, b) XylS and NahR; (c, d) XylS and HbpR; (e, f) NahR and HbpR, (g, h) XylS and DmpR, (i, j) NahR and DmpR, and (k, l) HbpR and DmpR. The experimental points and linear fitting curves of biosensor and its inducers are marked in different colors: XylS in red, NahR in green, HbpR in orange and DmpR in dark cyan.

Figure 5. Summary of the orthogonality among four sensors.