After obtaining these sensors’ coding sequence via PCR or synthesis, we constructed an expression system composed of two parts: (Fig 1)
(1) A constitutive Pc promoter linked with sensor’s coding sequence that encodes the regulating protein;
(2) The corresponding inducible promoter located in the front of RBS-sfGFP fluorescence reporter
We tested fluorescence intensity to show induction ratio of each expression system when exposed to their inducers through ELIZA and flow cytometry. Naturally, the performances of these transcriptional factors are not well characterized and needs further tuning to be biosensors.

Based on the data, we selectively changed Pc and RBS strength, tuning expression intensity of these transcriptional factors and sfGFP respectively, to optimize induction behavior.(Fig 2)

Up to now, we have constructed several well-performed types of aromatic biosensors including NahR, XylS, HbpR and DmpR. (Fig 3)

The well-characterized aromatic biosensors consist a comprehensive aromatic detection toolkit. Various aromatic compounds are involved in our toolkit’s detection range. The performance of these biosensors propose a possibility for pathway coupling, complex sample analysis and further band pass circuit application.

To apply the well-characterization biosensors we built in multicomponent analysis, the nonexistence of synergistic or antagonistic effects, in another word, orthogonality, among inducers should be confirmed.
We tested the orthogonality for all our fine-tuned biosensors. (please click here for further information) The result shows that within a general inducer concentration, the orthogonality of our biosensors fits the requirements of multi-components sample detection. (Fig. 4)

Fig 1. Schematic diagram of expression system
The transcriptional factor (TF) is ligated with Pc promoter on low-copy backbone pSB4K5. Reporter gene sfGFP is located downstream of promoter which is regulated by corresponding TF. The backbone is high-copy pUC57 simple. The dark purple arrowhead refers to Pc promoter, while the promoter regulated by corresponding TF is shown in cyan, dark green oval stands for Ribosome Binding Site (RBS), terminator is in dark red hexagon, dark blue square represents gene coding sequence.

Fig 2. Tuning on Pc promoter and RBS intensity
In order to obtain optimal induction performance, we constructed Pc promoter library, selecting J23106, J23105, J23114, J23117, J23109, J23113 and linked it with transcriptional factors. Besides, we constructed RBS library, adopting B0031, B0032, B0034 and put it upstream of reporter gene sfGFP. The arrowheads with blue gradient refer to different intensity of Pc promoter. The ovals with green gradient stand for distinct intensity of RBS.

Fig 3 well-performed aromatic biosensors and their detective range
Each color in the middle ring represents the detection range of a biosensor. Structural formula with color background stands for the aromatic compounds detected by our biosensors .┝ means plug in, connecting an enzyme with existing biosensor .

Fig. 4 Summary of the orthogonality between four sensors’ inducers. The inducers between XylS and NahR, XylS and HbpR, NahR and HbpR, XylS and DmpR, NahR and DmpR, and HbpR and DmpR are all highly orthogonal.