Next we focused on constructing biosensors with low basal level, high induction ratio, and robust detection profiles. The coding sequences of these 8 transcriptional regulators were obtained by either chemical synthesis or PCR amplification. They were then incorporated into our biosensor circuit design (Fig. 1):

It can be expected that the primary construction of a biosensor circuit might not work. For the fine-tuning, a library of constitutive promoters and a library of RBS sequences, both of different strength, were exploited to genetically tailor the expression of transcriptional regulators and sfGFP, respectively (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)

Figure 1. Schematic diagram of the biosensor circuit
A constitutive promoter (Pc) constitutively expresses the transcriptional regulator protein on the low-copy backbone pSB4K5; the cognate promoter of the transcriptional regulator controls the expression of the reporter gene, super-fold green fluorescent protein (sfGFP, a novel and robust GFP variant designed for in vivo measurement of protein expression levels); its backbone is high-copy pUC57. Blue arrow, Pc promoter; red arrow, the promoter regulated by the aromatics-sensing transcriptional regulator; dark green ovals, Ribosome Binding Sites (RBS); red hexagons, transcriptional terminators; orange squares, gene coding sequences.

Figure 2. Libraries of promoters and RBS sequences used for the fine-tuning of biosensor circuits.
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.