Based on the codon preference of E. coli, the QscR gene derived from Pseudomonas aeruginosa PAO1 was codon-optimized. Restriction sites (EcoRI, XbaI, SpeI, PstI) were eliminated to comply with BioBricks™ standard RFC#10 (verified via SnapGene® software). The complete genetic circuit, containing the J23100 constitutive promoter, B0034 ribosome binding site, optimized QscR gene, B0015 terminator, PQscR promoter, and mRFP reporter gene, was synthesized by Generalbial Company. This sequence was cloned into the pSB1A3 vector using XbaI and SpeI and transformed into E. coli BL21(DE3) competent cells (purchased from Beyotime) via heat shock (42°C, 1 min). Positive clones were selected on LB plates containing 50 µg/mL ampicillin. Single colonies were picked and inoculated into LB/Amp liquid medium, followed by shaking culture at 37°C until OD₆₀₀ reached 0.6-0.8. Preliminary verification was performed by colony PCR (using Vazyme 2×Taq Master Mix), and confirmation was obtained by sequencing (conducted by Tsingke Biotechnology Co., Beijing), ultimately yielding the engineered strain BL21-mRFP.
The sequence-verified engineered strain BL21-mRFP was inoculated into LB/Amp⁺ medium and cultured to mid-log phase (OD₆₀₀ ≈ 0.6). It was then mixed with an equal volume of 50% glycerol and stored at -20°C for future use.
3OC12-HSL (O9139), C10-HSL (07028), and 3OHC10-HSL (68873) purchased from Sigma-Aldrich were dissolved in DMSO to prepare 10 mM stock solutions. The constructed AHL biosensor engineered bacteria were inoculated at a 1:100 ratio into 5 mL of LB medium containing 50 µg/mL ampicillin and activated overnight at 37°C, 150 rpm. Then, 100 µL of the culture was transferred to 5 mL of fresh LB/Amp⁺ medium and cultured at 37°C, 180 rpm until OD₆₀₀ reached 0.3. Then, 1 mL of the bacterial culture was aliquoted into a 24-well plate, and 10 µM of 3OC12-HSL, C10-HSL, or 3OHC10-HSL was added separately. After continued cultivation for 3 hours at 37°C, 180 rpm, the absorbance at 600 nm (OD₆₀₀) and mRFP fluorescence intensity (excitation 584 nm/emission 607 nm) were simultaneously measured using a Molecular Devices FlexStation 3 microplate reader. The normalized fluorescence value (fluorescence intensity/OD₆₀₀) was calculated after subtracting the background value.
To test the dose-response effect, the AHL biosensor engineered bacteria were exposed to different concentrations of AHL molecules under the same culture conditions. The mRFP fluorescence response across different concentration gradients was detected using the Molecular Devices FlexStation 3 microplate reader to establish a dose-response curve.
The Indigo synthesis genes TnaA and FMO, codon-optimized to meet the RFC#10 standard, were synthesized. Based on the AHL biosensor, the mRFP reporter gene was replaced with the TnaA-B0034-FMO polycistronic unit. The J23100-B0034-QscR-B0015-PQscR-TnaA-B0034-FMO sequence was cloned into the pSB1A3 vector using XbaI and SpeI. The recombinant plasmid was transformed into E. coli BL21(DE3) competent cells (Beyotime) via heat shock. Positive clones were selected on LB plates containing 50 µg/mL ampicillin. The correct engineered strain was obtained after verification by colony PCR and sequencing (Tsingke, Beijing).
A single colony of the verified Indigo-producing engineered strain was inoculated into 5 mL of LB/Amp⁺ medium and activated overnight at 37°C. After centrifugation and washing, it was subcultured at a 1:100 ratio into 5 mL of M9 medium (Sangon Biotech) containing 50 µg/mL ampicillin, 0.4% glucose, 1 mM MgSO₄, and 50 µM CaCl₂. Different AHLs (3OC12-HSL, C10-HSL, or 3OHC10-HSL) were added to induce Indigo production. Then, 1 mL of bacterial culture was collected every 2 hours, centrifuged, and the supernatant was resuspended for absorbance measurement at 620 nm. A standard curve was prepared using Indigo standard (SM4168, Beyotime) for quantification. All experiments were performed with three replicates.
The salicylic acid synthesis genes ICS (derived from E. coli EntC) and IPL (derived from Pseudomonas fluorescens PchB), codon-optimized to meet the RFC#10 standard, were synthesized. The ICS-B0034-IPL sequence was cloned into the pET28a(m) vector using NdeI and XhoI restriction sites. The recombinant plasmid was transformed into E. coli BL21(DE3) competent cells (Beyotime). Positive clones were selected on LB plates containing 50 µg/mL kanamycin. The correct engineered strain was obtained after verification by colony PCR and sequencing (Tsingke, Beijing).
The verified salicylic acid engineered strain was inoculated at a 1:100 ratio into LB medium containing kanamycin and activated overnight at 37°C. Then, 100 µL of the culture was transferred to M9Y medium (M9 basal medium supplemented with 1 g/L yeast extract, 2 g/L MOPS, 10 g/L glycerol, 2.5 g/L glucose, 1 mM MgSO₄, and 50 µM CaCl₂) containing 50 µg/mL kanamycin. When the OD₆₀₀ reached 0.6 during cultivation at 37°C, 180 rpm, 0.5 mM IPTG was added to induce expression. Samples of 1 mL were taken every 24 hours, centrifuged, and the supernatant was collected. The salicylic acid concentration was detected using an ELISA kit, and the normalized yield (salicylic acid concentration/OD₆₀₀) was calculated.
Data analysis and graphing were performed using GraphPad Prism software. Data are presented as mean ± standard deviation (SD). For multiple group comparisons, one-way ANOVA with Tukey's post hoc test was used for difference analysis. For comparisons between two groups, Student's t-test was used. A p-value less than 0.05 was considered statistically significant.
