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 orthogonality between XylS, NahR, HbpR and DmpR biosensors have been carefully evaluated using the assay discussed above (Fig. 2). The data were processed by linear fitting and the slopes of the fitting curves were compared with 1 (Fig. 3, Fig. 4). The closer the slope was to 1, the more orthogonal the two biosensors were. Results showed that the biosensor pairs, 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 orthogonal, as 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; this allowed the combination of these biosensors to profile aromatics for the ease of practical applications. .

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. The orthogonality assay for the biosensor I and the biosensor II. (a) Biosensor I was added into the assay. Different inducer mixtures were added into lane 1, 2, and 3, respectively. 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 assay. Different mixtures of inducers were added into lane 1, 2, and 3, respectively. 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 perturbation of inducer B with the dose response of biosensor I to its inducer A, and vice versa. (a) The schematics for the orthogonality assay as in Fig. 2. (b) Horizontal axis represents the fluorescence when there is no inducer B perturbation and the vertical axis represents the fluorescence when the cell is exposed to the perturbation of inducer B in non-zero concentrations. If the dose response of biosensor I is constant, regardless of the concentrations of inducer B, the slope of the line should be close to 1, if not absolutely equal.

Figure 4. Linear fitting of the data obtained from the orthogonality assay showing that the orthogonality between the 4 representative biosensors. The experiments and data processing were performed as described in Fig. 2 and Fig. 3.The black dashed line denotes slope=1 as the reference line. These fittings showed the orthogonality between 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 experiment data, linear fitting curves of biosensor, and cognate inducers are in different colors: XylS in red, NahR in green, HbpR in orange and DmpR in dark cyan.

Figure 5. Summary of the orthogonality assay to evaluate the synergistic/antagonistic effects between the inducers of 4 representative biosensors. No synergistic or antagonistic effects between the inducers of 4 representative biosensors (XylS, NahR, HbpR, and DmpR) were observed. For instance, although the sensing profiles of NahR and XylS overlap to some extent, the NahR-specific and XylS-specific inducers proved to be really orthogonal.