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 strengths, were utilized to genetically tailor the expression of transcriptional regulators and sfGFP, respectively (Fig. 2).

After sparing no efforts to fine-tune the circuits, we have successfully constructed a comprehensive collection of high-performance aromatics-sening biosensors, including XylS, XylR, HbpR, HcaR, DmpR. and NahR. Results showed that a large variety of aromatic compounds have been taken into our toolkit’s detection profiles (Fig. 3). See the detailed information about the performance of Individual Biosensors.

To allow the combination of these biosensors to analyze aromatics profiles of practical samples, the synergistic and antagonistic effects between inducers, in another word, the orthogonality, should be carefully assessed. The results showed that, within a general range of inducer concentration, the orthogonality of our biosensors makes them effective at profiling practical samples (Fig. 4).

Figure 1. Schematic diagram for the design frame of biosensor circuits
A constitutive promoter (Pc) constitutively expresses the transcriptional regulator protein on the low-copy backbone pSB4K5; the cognate promoter of the transcriptional regulator (TF) 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. Left orange arrow, Pc promoter; right orange arrow, the promoter regulated by the aromatics-sensing transcriptional regulator; green ovals, Ribosome Binding Sites (RBS); red hexagons, transcriptional terminators; dark cyan squares, gene coding sequences.

Figure 2. Libraries of promoters and RBS sequences used for the fine-tuning of biosensor circuits.
Pc promoter library was exploited to fine-tune the expression level of transcriptional regulator: J23106, J23105, J23114, J23117, J23109 and J23113. The RBS library is composed of B0031, B0032, B0033 and B0034 to tailor the expression level of reporter gene sfGFP. Left orange arrow, Pc promoter; right orange arrow, the promoter regulated by the aromatics-sensing transcriptional regulator; green ovals, Ribosome Binding Sites (RBS); red hexagons, transcriptional terminators; dark cyan squares, gene coding sequences.

Figure 3. 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. The "plug" icon in the figure legend stand for Adaptors, enzymes that convert undetectable compounds into detectable aromatics, thus to expand the detection capacity of some biosensors. Click Here for the summary table of the aromatics spectrum and aromatics-sensing profiles of individual biosensors.

Figure 4. Summary of the orthogonality between the sensing-profiles of 4 representative biosensors.
The transcriptional regulator-specific inducer(s) of XylS, NahR, HbpR, and DmpR are all highly orthogonal to each other (e.g., although the sensing profiles of NahR and XylS overlap to some extent, in the orthogonality test we used NahR-specific and XylS-specific inducers and they proved to be really orthogonal), which is consistent with our expectation. Click Here to see the detailed information about the orthogonality tests.