We have equipped our toolkit with a number of individual biosensors, each capable of sensing a group of aromatic compounds (Fig 1). Considering the complexity of environmental samples, we may need to analyze a multi-component sample with 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 analyze both the concentration of 4-CISaA and the concentration of 3-CIBzO, we need to apply the biosensor NahR and XylS to the sample separately. In order to make sure of that the analyzing result is valid, we must insure that the existence of 3-CIBzO won’t interfere the dose-response of NahR to 4-CISaA, vice versa.

If the existence of each XylS’s inducer, which is not an inducer of NahR, doesn’t interfere the NahR’s dose-response to any of its inducers, vice versa, we call the biosensor NahR and XylS are orthogonal, and we are secured to apply the multi-component analysis with NahR and XylS.

We investigated the orthogonality between several pairs of biosensors(Fig.2). The orthogonality between biosensor I and biosensor II is tested in the following manner.

1. We choose a typical inducer A for biosensor I and a typical inducer B for biosensor II.
2. We measure the dose-response of biosensor I to inducer A under different concentration of inducer B.
3. We measure the dose-response of biosensor II to inducer B under different concentration of inducer A.

If biosensor I and biosensor II are orthogonal, the dose-response of biosensor I is invariant to the concentration of inducer B and the dose-response of biosensor II is invariant to the concentration of inducer A. This invariance is showed in the form of 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.