To address the serious threat of bacterial wilt caused by Ralstonia solanacearum in crops, we successfully developed a synthetic biology-based "detection-visualization-treatment" system through three complete "Design-Build-Test-Learn" (DBTL) cycles. This system consists of three core modules: the detection module constructs an AHL-responsive biosensor strain that can accurately report quorum sensing signals via red fluorescent protein (mRFP); the visualization module, through iterative improvement, successfully converts the signal into a visible blue color reaction; the treatment module, through rational design, constructs an engineered strain capable of efficiently synthesizing salicylic acid (SA), expected to activate the plant immune system and enhance resistance to bacterial wilt. After continuous optimization through three DBTL cycles, we successfully obtained all functional strains with stable and reliable performance, laying a solid foundation for the practical field application of the system.
The goal was to construct an engineered E. coli strain capable of specifically detecting acyl-homoserine lactone (AHL) quorum sensing signal molecules. Through literature review, we selected the quorum sensing regulatory protein QscR from Pseudomonas aeruginosa PAO1 and its native regulatory promoter PQscR as the core sensing elements. When QscR binds to AHL, it activates the expression of the downstream reporter gene mRFP, enabling quantitative detection of AHL presence and concentration through fluorescence intensity.
Figure 1 AHL Sensor Genetic Circuit
We first performed E. coli codon optimization on the QscR gene from P. aeruginosa PAO1 and eliminated internal XbaI, SpeI, EcoRI, and PstI restriction sites to ensure compliance with the BioBrick standard. The optimized gene sequence was synthesized by a professional biology company. Subsequently, the complete J23100-B0034-QscR-B0015-PQscR-mRFP expression cassette was precisely cloned into the standard pSB1A3 vector using XbaI and SpeI restriction enzymes, constructing the recombinant plasmid. This was transformed into E. coli BL21(DE3) cells, positive clones were verified by colony PCR, and finally the stable engineered strain named BL21-mRFP was obtained.
Figure 2 Construction of the AHL Biosensor Engineered Bacteria.
(A) AHL sensor recombinant plasmid map. (B) Agarose gel electrophoresis of QscR. (C) Agarose gel electrophoresis of mRFP.
The sequence-verified engineered strain was inoculated into LB medium and activated overnight, then subcultured at a 1:100 ratio into fresh LB medium. When the OD₆₀₀ reached 0.3 at 37°C, 1 mL of bacterial culture was transferred to a 24-well plate. AHL molecules including 10 μM 3OC12-HSL, C10-HSL, and 3OHC10-HSL were added separately. After 3 hours of induction at 37°C, the OD₆₀₀ value and mRFP fluorescence intensity at 584/607 nm were simultaneously detected using a microplate reader. After subtracting the background value, the normalized fluorescence value (fluorescence intensity/OD₆₀₀) for each sample was calculated, and the dose-response characteristics of the sensor were tested using different AHL concentrations.
The 3OC12-HSL induced group had the highest normalized fluorescence intensity (11.40 ± 0.52 RFU/OD), followed by the C10-HSL group (7.64 ± 0.31 RFU/OD), and the 3OHC10-HSL group had the weakest response (5.33 ± 0.16 RFU/OD).
Figure 3 Relative mRFP fluorescence intensity after 3h induction with different AHLs
The three AHL molecules showed weak responses in the 0.001-0.01 μM concentration range, a sharp increase in fluorescence intensity at 0.1 μM, and reached a response saturation plateau in the 1-10 μM concentration range. The sensor had the highest sensitivity to 3OC12-HSL (11.13 ± 0.53 RFU/OD), followed by C10-HSL (8.22 ± 0.49 RFU/OD) and 3OHC10-HSL (5.26 ± 0.45 RFU/OD).
Figure 4 Analysis of Biosensor Strain Sensitivity and Dose-Response Relationship
(A) Relative mRFP fluorescence intensity after 3h induction with different concentrations of 3OC12-HSL. (B) Relative mRFP fluorescence intensity after 3h induction with different concentrations of C10-HSL. (C) Relative mRFP fluorescence intensity after 3h induction with different concentrations of 3OHC10-HSL.
The experiment successfully verified that the QscR protein from P. aeruginosa PAO1 and its regulatory element PQscR can be efficiently assembled and function in the E. coli system. The constructed AHL biosensor engineered bacteria possess highly specific recognition capability for characteristic AHL molecules of R. solanacearum (3OC12-HSL, C10-HSL, 3OHC10-HSL), with the highest response sensitivity to 3OC12-HSL. However, preliminary application revealed that although the fluorescence-based reporter system performs excellently under laboratory conditions, its reliance on fluorescence detection equipment limits its promotion and application in field environments, making it particularly unsuitable for farmers lacking professional equipment.
Based on this finding, we systematically optimized the system by replacing the fluorescent reporter gene with the indigo synthesis pathway (TnaA and FMO), achieving a functional upgrade from instrument detection to visual observation. This improvement allows detection results to be directly interpreted via obvious blue precipitation, greatly enhancing the field applicability and user-friendliness of the system, laying a solid foundation for developing disease detection tools truly suitable for field environments.
To achieve visualization of detection, we upgraded the fluorescent reporter system in the already constructed AHL biosensor to a visible colorimetric output system. By replacing the reporter gene from mRFP with the indigo biosynthesis pathway genes (TnaA and FMO), we aimed to construct an engineered strain capable of producing blue indigo precipitate in response to AHL signals, enabling rapid, on-site visual detection without complex instruments.
Figure 5 indigo Colorimetric Biosensor Genetic Circuit
We first cloned the codon-optimized TnaA and FMO genes into the pSB1A3 vector using XbaI and SpeI restriction enzyme sites, replacing the original mRFP reporter gene, to construct the recombinant plasmid. The insertion of the TnaA and FMO genes was verified by colony PCR, and clones with correct PCR verification were sent for sequencing to confirm sequence accuracy. The sequenced, error-free engineered strain BL21-TnaA-FMO was cultured in LB/Amp⁺ medium to OD₆₀₀ ≈ 0.6, mixed with an equal volume of 50% glycerol, and stored at -80°C for backup.
Figure 6 Construction of AHL-Inducible indigo Biosynthesis Engineered Bacteria.
(A) indigo colorimetric biosensor plasmid map. (B) Agarose gel electrophoresis of TnaA. (C) Agarose gel electrophoresis of FMO.
The verified engineered strain was inoculated into M9 medium containing 0.4% glucose, and 1μM of 3OC12-HSL, C10-HSL, or 3OHC10-HSL was added to induce indigo synthesis. Then, 1 mL of bacterial culture was collected every 2 hours, centrifuged at 10,000 × g for 1 minute, and the supernatant was collected. The absorbance at 620 nm was measured using a microplate reader, using a 10 mM indigo solution dissolved in DMSO for calibration. A standard curve was prepared using different concentrations of indigo (0 mM, 0.25 mM, 0.5 mM, 1 mM, 1.5 mM, 3 mM). Each experimental group was performed in triplicate. indigo yield was quantitatively analyzed by comparison with the indigo standard curve. The experimental results showed that all three AHL molecules could effectively induce indigo synthesis. Within 0-2 hours, the indigo yield was very low; it began to accumulate significantly after 4 hours; explosive growth occurred at 6 hours; and within the 8-hour observation period, the yield reached its highest level. Among them, 3OC12-HSL had the strongest induction capability, with an 8-hour yield of 2.14 ± 0.14 mM, significantly higher than C10-HSL and 3OHC10-HSL. This time course indicates that to ensure reliable detection results, the observation time in field applications should not be less than 6 hours, with the optimal interpretation window being 6-8 hours.
Figure 7 Colorimetric Analysis of the indigo Biosensor.
(A) indigo standard curve. (B) Physical images of indigo production induced by different AHLs. (C) indigo content after treatment with 1μM 3OC12-HSL for different times. (D) indigo content after treatment with 1μM C10-HSL for different times. (E) indigo content after treatment with 1μM 3OHC10-HSL for different times.
This phase of research successfully achieved the technical upgrade of the AHL biosensor from fluorescence detection to visual detection. Through rational design, we replaced the reporter system from mRFP with the indigo biosynthesis pathway (TnaA-B0034-FMO), successfully constructing an engineered strain capable of producing visible blue precipitate in response to AHL signals. Based on achieving visual detection of AHL, a salicylic acid (SA) treatment system can be introduced to generate a visual detection signal (indigo precipitate) during the pathogen recognition stage, while continuously secreting salicylic acid, a plant systemic resistance activator, during the control stage, enhancing plant resistance to bacterial wilt.
Our goal was to construct an engineered strain capable of efficiently synthesizing salicylic acid (SA). We selected key genes for salicylic acid synthesis from plants and bacteria - isochorismate synthase gene (ICS) and isochorismate pyruvate lyase gene (IPL) - and ensured their efficient expression in E. coli through codon optimization. This engineered bacteria aims to enhance plant innate immunity against bacterial wilt through sustainable production of salicylic acid as a bioelicitor, providing a material basis for developing new biological control strategies.
Figure 8 Salicylic Acid Synthesis Engineered Bacteria Genetic Circuit
First, codon optimization for E. coli was performed on the key salicylic acid synthesis genes ICS and IPL, and the full genes were synthesized by a biology company. The optimized ICS-IPL fusion gene fragment was cloned into the corresponding multiple cloning site of the pET28a expression vector via NdeI and XhoI double digestion, constructing the recombinant expression plasmid pET28a-ICS-IPL. The verified correct recombinant plasmid was transformed into E. coli BL21(DE3) competent cells for positive clone screening. Single colonies were picked, the insertion of the target gene was verified by colony PCR, and after DNA sequencing confirmed the correct reading frame and absence of mutations, the recombinant engineered strain BL21/pET28a-ICS-IPL was obtained.
Figure 9 Salicylic Acid Synthesis Engineered Bacteria Plasmid Map
The verified engineered strain was inoculated into M9Y medium. When the OD₆₀₀ reached 0.6, 0.5 mM IPTG was added to induce protein expression. Cultivation continued, and 1 mL of bacterial culture was collected every 4 hours. After centrifugation at 10,000 × g for 1 minute, the supernatant was collected. The salicylic acid concentration in the supernatant was quantitatively analyzed using a salicylic acid ELISA detection kit, and the normalized yield was calculated. The results showed that after induction with 0.5 mM IPTG, the SA yield at 4h was 5.61 ± 1.55 mg/L, at 8h was 23.11 ± 2.91 mg/L, at 12h was 52.82 ± 3.31 mg/L, at 16h was 65.35 ± 6.29 mg/L, at 20h was 79.48 ± 6.67 mg/L, and at 24h was 84.02 ± 4.66 mg/L. After 24 hours of induction, the blank control group (BL21 empty strain) produced almost no salicylic acid (content close to 0 mg/L), while the engineered bacteria group (BL21-ICS-IPL) showed a significantly increased salicylic acid yield, exceeding 80 mg/L.
Figure 10 Salicylic Acid Production Test.
(A) Salicylic acid ELISA standard curve (A450). (B) Log10 values of salicylic acid concentration and A450 absorbance from ELISA analysis. (C) Salicylic acid production over time. (D) Salicylic acid content after 24h induction in different engineered strains.
This phase of research successfully constructed an engineered strain capable of efficiently synthesizing salicylic acid. Experimental verification showed that after IPTG induction, this strain can efficiently convert endogenous chorismate to salicylic acid, with a yield significantly higher than the blank control. The construction of this engineered bacteria provides a stable biosynthesis platform for studying the application of salicylic acid as a plant immune elicitor, achieving the functional expansion of the bacterial wilt control system from "detection" to "control".
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