Four chemically synthesized VOCs-responsive promoter fragments (PsoxS, PlasI, PrecA, and PgrpE) were cloned into the linearized pSB1A3 backbone using the ClonExpress® Ultra One Step Cloning Kit (Vazyme, China). The 10 μL reaction system contained ~100 ng linearized vector, equimolar amounts of insert DNA, 5 μL of 2× CE Ultra Mix, and sterile ddH₂O to final volume. The mixture was incubated at 37 °C for 30 min and cooled on ice. Each construct was designed to place the promoter upstream of the reporter gene mRFP.
The violacein biosynthetic gene cluster (vioABCDE) was digested with EcoRI-HF and XbaI (NEB, USA) at 37 °C for 1 h, together with the pSB1A3 vector. Digestion products were separated by 1% agarose gel electrophoresis, and target bands were purified using a Gel Extraction Kit (Omega Bio-tek, USA). The purified vector and insert were ligated overnight at 16 °C with T4 DNA ligase (NEB, USA) at a molar ratio of 1:3 (vector:insert).
The chitinase (RmChi44) and β-1,3-glucanase (MoGluB) genes were digested with NcoI and XhoI (NEB, USA) and cloned into identically digested pET28a(+) expression vector using the same digestion-ligation procedure. The constructs placed each gene downstream of the T7 promoter, introducing an N-terminal 6×His tag.
Each ligation mixture was transformed into 100 μL chemically competent E. coli DH5α cells (Qingke Bio, China). Cells were incubated on ice for 30 min, heat-shocked at 42 °C for 45 s, and cooled on ice for 2 min. Then, 900 μL antibiotic-free LB broth was added, and cultures were recovered at 37 °C and 220 rpm for 1 h. Appropriate volumes were plated on LB agar containing the corresponding antibiotics (pSB1A3 constructs: 100 μg/mL ampicillin; pET28a constructs: 50 μg/mL kanamycin) and incubated overnight at 37 °C.
Single colonies were screened by colony PCR using vector-specific or gene-specific primers. Positive clones were cultured overnight in 5 mL LB with the corresponding antibiotic. Plasmids were extracted using a Miniprep Kit (Tiangen Biotech, China), verified by double digestion, and confirmed by Sanger sequencing (Qingke Biotechnology, Beijing). Verified pSB1A3 constructs were transformed into E. coli BL21(DE3) competent cells using the same procedure. All validated recombinant strains were mixed 1:1 with sterile 60% glycerol and stored at −80 °C.
Glycerol stocks of the four reporter strains [BL21(DE3)/pSB1A3-PsoxS-mRFP, etc.] were streaked onto LB agar plates containing 100 μg/mL ampicillin and incubated at 37 °C for 16 h. A single colony was inoculated into 5 mL of LB medium with the same antibiotic and cultured overnight (37 °C, 250 rpm, ~16 h). The overnight culture was diluted 1:100 into 10 mL fresh LB in 50 mL flasks and grown under the same conditions until OD₆₀₀ = 0.6 ± 0.05.
VOCs stock solutions (1-octanol, 1-octen-3-ol, and phenylethyl alcohol, ≥ 98%, Sigma-Aldrich) were first diluted in sterile deionized water to 0.1% (v/v), then further diluted 1,000× before use. Ninety microliters of bacterial culture was added per well into a 96-well black clear-bottom plate (Corning, USA), followed by 10 μL of the diluted VOCs solution (final volume = 100 μL). Control wells received 10 μL sterile deionized water. The plate was sealed with a breathable film and incubated statically at 37 °C for 4 h.
After incubation, the plate was gently shaken for 10 s, and OD₆₀₀ was measured to estimate cell density using a FlexStation 3 multi-mode microplate reader (Molecular Devices, USA). mRFP fluorescence was recorded at 584 nm excitation and 607 nm emission. Each sample was measured in triplicate.
Normalized fluorescence was calculated as RFU/OD₆₀₀. Fold change = (mean normalized fluorescence of VOCs-treated group) / (mean normalized fluorescence of control group). Each experiment was independently repeated three times (n = 3, with triplicate technical replicates). Data were presented as mean ± SD, analyzed by one-way ANOVA with Tukey’s multiple comparison test (p < 0.05 considered significant) using GraphPad Prism 9.0.
The violacein-producing strain [BL21(DE3)/pSB1A3-J23100-vioABCDE] was inoculated into M9 minimal medium containing 100 μg/mL ampicillin (50 mL per 250 mL flask), supplemented with either 5% (w/v) glucose or 5% (v/v) glycerol as carbon source. Cultures were incubated at 37 °C and 220 rpm. Samples (5 mL) were taken aseptically at predetermined time points (1, 3, 8, 12, 24, 36, 48, 60 h).
Two milliliters of culture was centrifuged at 12,000 × g for 10 min at 4 °C. The supernatant and cell pellet were separated. The pellet was washed once with 1 mL prechilled PBS (pH 7.4), centrifuged again, and resuspended in 1 mL absolute ethanol. Cell suspensions were sonicated in an ice bath using a probe sonicator (Ningbo Xinzhi, China) at 300 W, 3 s on/5 s off, for 10 min total. The lysate was centrifuged again (12,000 × g, 10 min). Meanwhile, 1 mL of culture supernatant was mixed with 1 mL ethanol and centrifuged similarly. Ethanol extracts from both supernatant and lysate were combined for quantification.
Absorbance at 575 nm was measured using a UV–Vis spectrophotometer (Shimadzu, Japan). Violacein concentration was determined using a standard curve prepared with violacein standard (Sigma-Aldrich) dissolved in 50% ethanol at 0, 2.5, 5, 10, 15, 20 μg/mL. The biomass concentration was determined from OD₆₀₀ using a pre-established OD₆₀₀–DCW (dry cell weight) correlation.
Recombinant strains [BL21(DE3)/pET28a-Chitinase or pET28a-Glucanase] were cultured overnight in 10 mL LB with 50 μg/mL kanamycin at 37 °C, 220 rpm. The following day, 1% of the overnight culture was inoculated into 200 mL fresh LB (1 L flask) and grown at 250 rpm until OD₆₀₀ = 0.6–0.8. IPTG (Sigma-Aldrich) was added to a final concentration of 1 mM, and incubation was continued at 30 °C for 12 h. Cells were harvested by centrifugation (8,000 × g, 10 min, 4 °C) and resuspended in 20 mL binding buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0).
Cells were lysed by sonication (400 W, 5 s on/10 s off, 30 min) in an ice bath. The lysate was centrifuged (12,000 × g, 30 min, 4 °C), and the supernatant was applied to a Ni–NTA affinity column (Qiagen, Germany) pre-equilibrated with binding buffer at 1 mL/min. The column was washed with buffer containing 40 mM and 60 mM imidazole and eluted with 250 mM imidazole. Eluates were dialyzed overnight against PBS (pH 7.4) at 4 °C to remove imidazole. Protein concentrations were determined using a BCA Protein Assay Kit (Beyotime, China). Ten micrograms of purified protein was analyzed by 12% SDS–PAGE and stained with Coomassie Brilliant Blue R-250 to verify molecular weight and purity.
Saccharomyces cerevisiae BY4741 was streaked on YPD agar (1% yeast extract, 2% peptone, 2% glucose, 2% agar) and incubated at 30 °C for 48 h. A single colony was inoculated into 5 mL YPD broth and grown at 30 °C, 200 rpm for 16 h to logarithmic phase. Cells were washed twice with sterile PBS (pH 6.0) and adjusted to 1 × 10⁶ CFU/mL (counted by hemocytometer and verified by OD₆₀₀–CFU calibration).
The following reaction groups were prepared in 1.5 mL tubes (final volume = 200 μL):
(1)
(2)
(3)
(4)
All mixtures were incubated at 30 °C, 100 rpm for 2 h. After incubation, mixtures were serially diluted 10-fold (10⁻¹–10⁻⁴) in PBS, and 100 μL from each dilution was spread on YPD agar plates. Plates were incubated at 30 °C for 36–48 h, and colonies were counted. Each group included three biological replicates and two technical replicates.
Relative viability (%) = (average CFU/mL of experimental group / average CFU/mL of control) × 100%. Data were expressed as mean ± SD and analyzed by one-way ANOVA.
Design primers with ≤ 2 °C Tm difference and minimal dimers/hairpins. For inhibitor-rich samples, use purified DNA or inhibitor-resistant polymerases. Use colony PCR for screening transformants.
Pick single colonies with a sterile toothpick or pipette tip, streak briefly on selective LB plate (for preservation), then dip into 20 μL PCR mix or suspend in 20 μL ddH₂O and boil (95 °C, 5 min) before using 1 μL as template.
Run PCR with 30–35 cycles; analyze amplicons by agarose gel electrophoresis.
Use 0.8% agarose for > 1 kb fragments, 1.0% for 0.5–5 kb, and 1.5–2.0% for < 1 kb. Prepare gels in 1× TAE or 1× TBE buffer with DNA dye (e.g., GelRed).
Run at 5–8 V/cm (typically 100 V for 30–60 min). Visualize bands briefly under low-intensity UV. Excise target bands and purify with a gel extraction kit, eluting in 30–50 μL. Verify DNA quality before ligation or sequencing.
Digest vector and insert according to enzyme specifications (typically 37 °C, 1 h; overnight for complete digestion).
Dephosphorylate vectors with CIP/FastAP if necessary.
Use 1:3 vector:insert molar ratio.
T4 DNA ligase reactions were performed at 16 °C overnight or RT for 10–30 min, or assembled by seamless cloning (e.g., Gibson/ClonExpress) per manufacturer instructions.
Extract plasmids using commercial alkaline lysis kits following manufacturer protocols. Ensure complete lysis, proper neutralization, and thorough washing. Elute DNA in 30–50 μL EB buffer or ddH₂O.
Check plasmid quality by restriction digestion and spectrophotometry (A260/A280 ≈ 1.8–2.0; A260/A230 > 1.8). Adjust plasmid concentration to 50–100 ng/μL for sequencing.
