Experimental Record of the Strawberry Spoilage Detection System and Enzyme-Based Sterilization System

Team Members: Luo Youran, Wang Lu, Fan Yuchen, Lai Yuxin, Yang Ziyao, Huang Zhenqing, Jin Mingzhen, Li Yuhan, Feng Sihui, Wen Ying, Feng Anbo, Hu Yanglin, Xu Haoxuan, Pan Yi, Du Yuxin, Lu Zihan, Chen Junyu, Huang Xianyang, Ma Xinran, Han Hanhui, Sun Xiaoya, He Daixi, Wang Tianyu, Liu Jiayi, Liu Shuya, Cai Shiru, Yang Jiaqi

Experimental Period: July 8, 2024 – September 5, 2024

Abstract:

This experimental record details the construction and validation processes of the core modules for the project concerning the Strawberry Spoilage Detection System and Enzyme-Based Sterilization System. The primary objectives encompassed: 1) Screening for the optimal promoter responsive to characteristic volatile organic compounds (VOCs) associated with strawberry spoilage; 2) Constructing and optimizing a violacein-based visible light reporter system; and 3) Expressing and validating the antifungal activity of chitinase and glucanase. The experiments successfully identified the PgrpE promoter as the most effective VOC-responsive element, demonstrated that glycerol significantly enhances violacein production, and yielded highly purified antifungal enzymes. These outcomes establish a solid foundation for the subsequent development of the integrated system.

Phase 1: Construction and Validation of the VOC-Responsive Promoter Screening System (July 8 – August 2)

July 8 – Experimental Preparation and Strain Activation

Experimental Principle:

Strain activation involves transferring cryopreserved bacterial stocks into fresh culture medium to restore normal growth and metabolic activity, thereby providing experimental materials with consistent viability and physiological state for subsequent procedures. LB medium (Luria-Bertani medium) supplies essential nutrients, including a carbon source (tryptone), a nitrogen source (yeast extract), vitamins, and minerals (sodium chloride). Aseptic technique is critical to prevent microbial contamination and ensure the reliability of experimental results.

Experimental Methods:

1. LB Medium Preparation:
2. Liquid LB: Tryptone (10 g), yeast extract (5 g), and NaCl (10 g) were dissolved in 1 L of deionized water with stirring until complete dissolution. The solution was aliquoted into conical flasks.
3. Solid LB: Agar (15 g) was added to the 1 L liquid LB formulation described above.
4. Both media were autoclaved at 121°C for 20 minutes.
5. Antibiotic Plate Preparation:After sterilization, the solid LB medium was cooled to approximately 50-55°C (perceived as hot but tolerable to the back of the hand). Within a laminar flow hood, ampicillin stock solution (100 mg/mL) was added to a final concentration of 100 μg/mL (i.e., 100 μL per 100 mL of medium). The flask was gently swirled to ensure mixing while minimizing bubble formation. The medium was promptly poured into sterile Petri dishes (approximately 15-20 mL per dish). The plates were covered, left undisturbed until the agar solidified completely, then inverted, sealed with parafilm, and stored at 4°C.
6. Strain Activation:
7. DH5α strains (individually harboring the plasmids pSB1A3-PsoxS-mRFP, -PlasI-mRFP, -PrecA-mRFP, -PgrpE-mRFP) and BL21(DE3) strains were retrieved from the -80°C freezer and immediately thawed on ice for 10-15 minutes.
8. The laminar flow hood was irradiated with UV light for 30 minutes for surface sterilization. The UV light was then turned off, and the fan and light were switched on. Hands and the work surface were wiped with 75% ethanol.
9. An alcohol lamp was ignited to create a sterile zone. Sterilized LB plates and sterile inoculation loops were placed inside the hood.
10. Using a cooled inoculation loop, a small amount of bacterial suspension was streaked onto the surface of Amp-LB plates using the quadrant streak method to obtain isolated single colonies.
11. The plates were inverted and incubated in a 37°C constant-temperature incubator for 16-18 hours.

Results:

Well-dispersed single colonies appeared on all plates after incubation, indicating successful strain activation without contamination.

Experimental Reflection:

This initial experience operating the laminar flow hood provided a deeper understanding of key aspects of aseptic technique, such as working near the alcohol lamp flame and proper placement of items. It reinforced the necessity of strict sterilization for all utensils contacting samples, as any oversight could lead to contamination in subsequent experiments. It was noted that colony morphologies varied slightly between strains containing different plasmids, but all variations fell within the normal expected range.

July 9 – Plasmid Extraction and PCR Verification

Experimental Principles:

• Plasmid Extraction (Alkaline Lysis Method): This method utilizes alkaline conditions (SDS/NaOH) to lyse bacterial cells, denaturing chromosomal DNA and proteins, which precipitate as a viscous complex. Due to its supercoiled conformation, plasmid DNA remains soluble. Neutralization with an acidic potassium acetate solution promotes more complete precipitation. Following centrifugation, the plasmid DNA is recovered in the supernatant. Subsequent purification via a silica membrane column (where DNA binds under high-salt conditions and is eluted with a low-salt buffer or water) yields high-purity plasmid DNA.
• Polymerase Chain Reaction (PCR): PCR employs DNA polymerase to specifically amplify target DNA fragments in vitro. This is achieved through repeated cycles (typically 25-35) of three steps: denaturation (high temperature, ~95°C, to separate DNA strands), annealing (lower temperature, 55-65°C, allowing primers to bind specifically to the template), and extension (medium temperature, ~72°C, where DNA polymerase synthesizes new strands). This process results in exponential amplification of the target sequence.

Experimental Methods:

1. Shake-Flask Culture: Single colonies of the four recombinant plasmids from the previous day's activation plates were picked using sterile toothpicks or pipette tips and inoculated into four tubes containing 5 mL of LB liquid medium (with Amp 100 µg/mL). Cultures were incubated at 37°C with shaking at 220 rpm for 14-16 hours.
2. Plasmid Extraction (Using Omega Bio-Tek Plasmid Mini Kit I):
3. 1.5-5 mL of bacterial culture (volume adjusted based on culture density) was transferred to a microcentrifuge tube and centrifuged at 12,000 rpm for 1 minute. The supernatant was completely discarded.
4. The pellet was thoroughly resuspended by vortexing in 250 µL of Buffer P1 (containing RNase A).
5. 250 µL of Buffer P2 was added, and the tube was inverted gently 6-8 times to lyse the cells completely, resulting in a clear, viscous solution. This was incubated at room temperature for no more than 5 minutes.
6. 350 µL of Buffer N3 was added, and the tube was inverted immediately and gently 6-8 times, yielding a white flocculent precipitate. The sample was centrifuged at 12,000 rpm for 10 minutes.
7. The supernatant was carefully transferred to a HiBind® DNA Mini Column assembled in a collection tube, avoiding transfer of any precipitate. It was centrifuged at 12,000 rpm for 1 minute, and the flow-through was discarded.
8. 500 µL of Buffer HB was added to the column, centrifuged at 12,000 rpm for 1 minute, and the flow-through was discarded.
9. 700 µL of DNA Wash Buffer (with ethanol) was added to the column, centrifuged at 12,000 rpm for 1 minute, and the flow-through was discarded. This wash step was repeated once.
10. The empty column was centrifuged at 12,000 rpm for 2 minutes to remove residual ethanol completely.
11. The column was placed in a new 1.5 mL microcentrifuge tube. 30-50 µL of Elution Buffer or sterile water (pre-warmed to 65°C can increase elution efficiency) was added to the center of the membrane, followed by incubation at room temperature for 2 minutes. DNA was eluted by centrifugation at 12,000 rpm for 1 minute.

1. Concentration and Purity Determination: Plasmid concentration and purity were measured using a NanoDrop 2000 spectrophotometer. After zeroing with Elution Buffer, 1-2 µL of plasmid solution was measured, and the concentration (ng/µL) and A260/A280 ratio (ideal range 1.8-2.0) were recorded.
2. Colony PCR Verification:
3. Reaction Mixture (25 µL):
4. 2× Taq PCR MasterMix (with dye): 12.5 µL
5. Forward Primer (10 µM): 1 µL
6. Reverse Primer (10 µM): 1 µL
7. Template DNA (colony): A small amount of a single colony from the plate to be verified was touched with a sterile toothpick and stirred into the PCR mix.
8. ddH₂O: Added to a final volume of 25 µL.
9. Thermal Cycler Program (Bio-Rad T100):
10. Initial Denaturation: 95°C for 5 min
11. Cycling (30 cycles): 95°C for 30 s, 58°C for 30 s, 72°C for 1 min/kb
12. Final Extension: 72°C for 5 min
13. Hold: 4°C
14. Agarose Gel Electrophoresis:
15. 0.36 g of agarose powder was added to 30 mL of 1× TAE electrophoresis buffer and heated in a microwave until completely dissolved and clear.
16. After cooling to approximately 60°C, 3 µL of GelRed nucleic acid dye was added, mixed, and poured into a gel tray with a comb. The gel was solidified at room temperature for 30-40 minutes.
17. After solidification, the comb was removed, and the gel was placed in the electrophoresis tank submerged in 1× TAE buffer.
18. 5 µL of PCR product was mixed with 1 µL of 6× DNA Loading Buffer and carefully loaded into the wells. 5 µL of DL2000 DNA Marker was loaded into an adjacent well.
19. Electrophoresis was performed at a constant voltage of 120V for 25-30 minutes, until the bromophenol blue dye had migrated approximately two-thirds the length of the gel.
20. The gel was visualized and photographed under UV light using a gel documentation system (Bio-Rad ChemiDoc™).

Results and Analysis:

• Plasmid Concentration Determination: The concentrations of the four extracted plasmids ranged between 80-150 ng/µL, with A260/A280 ratios around 1.85, indicating good purity.
• Agarose Gel Electrophoresis: PCR products showed single, clear bands corresponding to the expected sizes (mRFP ~678 bp, PsoxS-mRFP ~740 bp, PlasI-mRFP ~835 bp, PrecA-mRFP ~802 bp, PgrpE-mRFP ~776 bp).

Conclusion:

Plasmids were successfully extracted from the colonies, and PCR verification confirmed the presence of the four "promoter-mRFP" reporter plasmids in their respective strains, indicating correct construction.

Experimental Incident and Reflection:

• Incident: For one of the PrecA-mRFP colonies, the PCR product showed an additional, non-specific band below the main band on the gel.
• Solution: This was hypothesized to be due to primer-dimer formation or non-specific primer annealing. A gradient PCR with varying annealing temperatures (56°C, 58°C, 60°C) was performed. It was found that increasing the annealing temperature to 60°C eliminated the non-specific band while maintaining a clear main band. Subsequent verifications utilized a 60°C annealing temperature.
• Reflection: PCR conditions require optimization for different primer pairs and cannot be universally applied. The inclusion of appropriate controls—a positive control (a plasmid with a known correct sequence) and a negative control (water as template)—is crucial. These controls help determine if results originate from the intended template or from contamination. In this experiment, a negative control was included and yielded no product, effectively ruling out contamination.

July 10 – Preparation of Competent Cells and Plasmid Transformation

Experimental Principle:

The calcium chloride (CaCl₂) method for preparing competent cells utilizes treatment with Ca²⁺ ions at low temperatures (0-4°C) to alter cell membrane permeability, inducing a physiological "competent" state that facilitates the uptake of exogenous DNA. A subsequent 42°C heat shock briefly increases membrane fluidity (heat shock effect), promoting the entry of DNA molecules into the cells. A recovery period in a nutrient-rich, antibiotic-free medium allows the cells to repair and express the plasmid-borne antibiotic resistance gene, enabling growth on antibiotic-containing plates.

Experimental Methods:

1. Preparation of Competent Cells (CaCl₂ Method):

• A single colony of BL21(DE3) from a freshly streaked plate was inoculated into 5 mL of LB medium and incubated overnight at 37°C with shaking at 220 rpm.
• 1 mL of the overnight culture was transferred into 100 mL of LB medium (in a 500 mL conical flask) at a 1:100 dilution ratio. The culture was incubated at 37°C with vigorous shaking at 220 rpm until an OD600 of approximately 0.4-0.5 was reached (approximately 2-3 hours, requiring periodic OD monitoring).
• The culture was cooled on ice for 30 minutes. The cells were then harvested by centrifugation at 4°C and 5,000 × g for 10 minutes.
• The supernatant was discarded, and the cell pellet was gently resuspended in 10 mL of ice-cold, sterile 0.1 M CaCl₂ solution (avoiding vigorous vortexing). The suspension was incubated on ice for 30 minutes.
• The cells were pelleted again by centrifugation at 4°C and 5,000 × g for 10 minutes, and the supernatant was discarded.
• The pellet was gently resuspended in 2 mL of ice-cold 0.1 M CaCl₂ solution containing 15% glycerol. The suspension was aliquoted into 100 μL volumes per tube and rapidly frozen at -80°C for storage.

2. Heat-Shock Transformation:

• Prepared BL21(DE3) competent cells were retrieved from -80°C and thawed on ice.
• Approximately 100 ng (typically 1-5 μL) of plasmid DNA (PsoxS-mRFP, PlasI-mRFP, PrecA-mRFP, PgrpE-mRFP) was added to 100 μL of competent cells and mixed gently by pipetting. A negative control tube without plasmid DNA was prepared.
• The mixture was incubated on ice for 30 minutes.
• The tubes were subjected to a heat shock by floating them in a 42°C water bath for exactly 90 seconds.
• The tubes were immediately transferred back to ice for a 2-3 minute cooling period.
• 900 μL of LB medium without antibiotics was added to each tube. The cells were allowed to recover by incubation at 37°C with slow shaking at 150 rpm for 45 minutes.
• The transformation culture was centrifuged at 4°C and 5,000 × g for 3 minutes. Most of the supernatant was carefully removed, leaving approximately 100 μL of medium. The cell pellet was gently resuspended in the remaining liquid by pipetting.
• The entire resuspended culture was spread onto pre-warmed Amp-LB plates and evenly distributed using a sterile spreader.
• After the liquid on the plate surface was absorbed, the plates were inverted and incubated at 37°C for 12-16 hours.

Results:

Colonies grew on all plates from the transformation groups, whereas no growth was observed on the negative control plate. This indicates the successful transformation of all four recombinant plasmids into the BL21(DE3) host strain.

Experimental Reflection:

• Maintaining low temperatures throughout the procedure is critical; all solutions and tubes must be pre-chilled, as temperature fluctuations at any step can significantly reduce transformation efficiency.
• The duration of the heat shock must be precise; excessive time can kill the cells, while insufficient time results in low efficiency.
• The recovery step is essential, as it provides time for cellular repair and expression of the antibiotic resistance gene. Concentrating the cells by centrifugation before plating increases the likelihood of obtaining isolated single colonies.

July 11 – August 2 – VOC Response Experiments and Promoter Screening

Experimental Principle:

Certain bacterial promoters can be activated by specific environmental signals, such as oxidative stress or membrane damage. Volatile organic compounds (VOCs) produced during strawberry spoilage (e.g., alcohols, esters) may act as stress signals, eliciting a stress response in E. coli. We constructed biosensors by linking stress-responsive promoters—PsoxS (oxidative stress), PlasI (quorum sensing/membrane damage), PrecA (SOS response/DNA damage), and PgrpE (heat shock/protein misfolding)—to the reporter gene mRFP (red fluorescent protein). Upon VOC exposure, promoter activation drives mRFP expression, with fluorescence intensity proportional to VOC concentration/intensity. By simultaneously measuring fluorescence and cell density (OD600) and calculating the fluorescence-to-OD600 ratio, we normalized for cell density variations, allowing quantitative comparison of promoter sensitivity to specific VOCs, typically expressed as the induction fold change.

Experimental Methods:

1. Strain Preparation: Single colonies of BL21(DE3) harboring the four reporter plasmids were picked from transformation plates and inoculated into 5 mL of LB medium containing Amp. Cultures were grown overnight at 37°C with shaking at 220 rpm.
2. VOC Stock and Working Solution Preparation:
3. Stock Solutions: 1 M stock solutions of 1-octanol, 1-octen-3-ol, and phenylethyl alcohol were prepared in dimethyl sulfoxide (DMSO) and stored at -20°C.
4. Working Solutions: On the day of the experiment, stock solutions were diluted 100-fold in sterile deionized water to yield 10 mM working solutions. The final concentration of DMSO was kept consistent (<1% v/v) across all experimental groups.
5. 96-Well Plate Induction Assay:
6. Overnight cultures were diluted 1:100 into fresh LB medium (with Amp) and grown at 37°C to mid-log phase (OD600 ≈ 0.5-0.6).
7. 90 μL of bacterial culture was aliquoted into each well of a sterile, black-walled, clear-bottom 96-well plate (Costar®).
8. Experimental Groups: 10 μL of a specific 10 mM VOC working solution was added to respective wells (final concentration: 1 mM).
9. Solvent Control Group (CK-): 10 μL of sterile water containing 1% DMSO was added.
10. Blank Control Group: 100 μL of sterile LB medium was used.
11. Each strain-treatment combination was performed in triplicate (three biological replicates).
12. The plate was sealed with a breathable membrane to prevent evaporation and contamination.
13. The plate was incubated statically for 4 hours either in a temperature-controlled microplate shaker/reader (e.g., Tecan Spark®) or in a 37°C incubator.

1. Detection:
2. After incubation, the plate was placed in the microplate reader.
3. OD600 was measured first (pathlength setting: automatic or fixed, e.g., 5.5 mm).
4. mRFP fluorescence intensity was then measured using the following parameters: Excitation = 584 nm, Emission = 607 nm, Bandwidth = 10 nm, Gain = optimized in pre-experiments (e.g., 150), using bottom fluorescence reading mode.
5. Data Processing:
6. Normalized fluorescence for each well was calculated: Normalized Fluorescence = (Fluorescencesample - Fluorescenceblank) / (OD600sample - OD600blank).
7. The average normalized fluorescence was calculated for each experimental group (n=3).
8. The fold change (FC) was calculated: FC = Average Normalized Fluorescenceexperimental group / Average Normalized Fluorescencesolvent control group.

Results and Analysis:

• PgrpE Time-Course Response: The PgrpE engineered strain was induced with a mixture of VOCs (phenylethyl alcohol : 1-octanol : 1-octen-3-ol = 6:1:1, total concentration 1 mM). Samples were taken at 0, 2, 4, 6, and 12 hours. Results showed a detectable response at 2 hours (FC ≈ 1.5), which increased to 2.8-fold at 4 hours, peaked at 3.5-fold at 6 hours, and subsequently decreased slightly but remained above 3.0-fold.

Conclusion:

Based on a comprehensive evaluation of response intensity, stability, and broad-spectrum responsiveness to multiple VOCs, the PgrpE promoter was identified as the most sensitive and stable core sensing element for the target strawberry spoilage VOCs. It was selected for subsequent construction of the integrated detection system.

Experimental Incidents and Reflections:

• Incident 1: Initial experiments revealed abnormally high and variable fluorescence background even in the solvent control (CK-) group.
• Solution and Reflection 1: Investigation identified two causes: 1) The LB medium used had been sterilized days prior and stored at 4°C, potentially leading to the generation of auto-fluorescent compounds or compositional changes. 2) Evaporation from edge wells in the 96-well plate caused volume and concentration changes, creating an "edge effect." We subsequently used LB medium prepared and sterilized on the same day and, during formal experiments, loaded samples only in the inner wells of the plate, filling the outer wells with PBS buffer or water to maintain humidity. These measures significantly reduced background noise and improved data consistency and reliability.
• Incident 2: When testing the response of PsoxS to 1-octanol, significant growth inhibition (substantially lower OD600 compared to the control) was observed at high concentrations (e.g., 2 mM).
• Solution and Reflection 2: This indicated potential cytotoxicity of the VOCs themselves at high concentrations. Consequently, we performed VOC concentration gradient experiments (0.1, 0.5, 1.0 mM) and ultimately selected 1 mM as the working concentration. This concentration elicited a significant response without severely inhibiting cell growth. This highlights the necessity of balancing signal intensity with host cell viability in biosensor applications.

Overall Reflection:

1. The quality and freshness of the culture medium profoundly impact fluorescence-based assays. 2) Edge effects must be considered and mitigated in microplate experiments. 3) Data normalization (Fluorescence/OD600) is crucial for eliminating the influence of cell density variations and is more scientifically robust than relying solely on absolute fluorescence values. 4) The inclusion of stringent controls (blank, solvent control) is fundamental for obtaining credible results.

Phase 2: Construction and Optimization of the Violacein Reporter System (August 5 – August 20)

August 5 – Establishment of the Violacein Standard Curve

Experimental Principle:

According to the Lambert-Beer Law, within a certain concentration range, the absorbance (A) of a solution for monochromatic light is proportional to its concentration (C) and the path length (b) of the light through the solution, expressed as A = εbC (where ε is the molar absorptivity). By measuring the absorbance of a series of standard solutions with known concentrations and plotting a standard curve (A vs. C), the concentration of an unknown sample can be determined from its measured absorbance using the regression equation of the standard curve.

Experimental Methods:

1. Preparation of Violacein Standard Stock Solution: Precisely 1.0 mg of violacein standard (Sigma-Aldrich) was dissolved in 1 mL of absolute ethanol to prepare a 1 mg/mL (1000 μg/mL) stock solution. This stock was stored protected from light at -20°C.
2. Preparation of Standard Series Solutions: Six 2 mL microcentrifuge tubes were used. The stock solution was diluted with 50% (v/v) aqueous ethanol solution according to a predefined dilution scheme (table implied but not fully detailed in the original text) to create a concentration series.
3. Full Wavelength Scan: A 10 μg/mL standard solution was scanned from 300 nm to 700 nm using a UV-Vis spectrophotometer (Shimadzu UV-1800), with 50% ethanol as the reference, to determine the maximum absorption wavelength (λmax).
4. Absorbance Measurement and Standard Curve Plotting: At the determined λmax wavelength, the spectrophotometer was zeroed using 50% ethanol. The absorbance (A) of each standard solution in the series was then measured sequentially. Each concentration was measured in triplicate, and the average value was calculated. Concentration (μg/mL) was plotted on the X-axis and the average absorbance value (A) on the Y-axis. Linear regression analysis was performed using OriginPro software to obtain the standard curve equation Y = aX + b and the correlation coefficient (R²).

Results and Analysis:

• The full wavelength scan indicated that the maximum absorption wavelength (λmax) for violacein in 50% ethanol is 575 nm.

concentration (μg/mL)	0	2.5	5	10	15	20
Absorbance (A575 nm)	0.002	0.151	0.298	0.612	0.925	1.234

• The linear regression equation for the standard curve was: Y = 0.0618X - 0.0045, with a correlation coefficient R² = 0.9996. This demonstrates an excellent linear relationship within the 0-20 μg/mL concentration range.

Experimental Reflection:

The standard curve must be applied strictly within its established linear range. We confirmed the excellent linearity within the 0-20 μg/mL range. To ensure accuracy and account for potential instrument drift, it is recommended to re-measure one or two points from the standard curve (e.g., 0 and 10 μg/mL) each time unknown samples are assayed. Standard solutions must be stored protected from light to prevent degradation.

August 6-12 – Construction of the Violacein-Producing Engineered Strain and Fermentation

Experimental Principle:

Violacein is a purple indole pigment synthesized via an enzymatic pathway encoded by the chromosomal Vio operon (containing genes vioA, vioB, vioC, vioD, and vioE). For heterologous expression in E. coli BL21(DE3), the codon-optimized complete vioABDCE gene cluster was cloned into the pSB1A3 plasmid, driven by the constitutive strong promoter PJ23100 and the strong ribosome binding site (RBS) B0034. The pigment is not secreted and primarily accumulates intracellularly. It can be extracted using organic solvents (e.g., ethanol, DMSO) to disrupt the cell wall and membrane.

Experimental Methods:

1. Engineered Strain Construction: An expression cassette containing PJ23100-B0034-vioABDCE was synthesized by a commercial vendor and cloned between the EcoRI and PstI sites of the pSB1A3 vector. This recombinant plasmid was transformed into E. coli BL21(DE3) via heat shock. Positive clones were selected on Amp-LB plates and verified by colony PCR and sequencing.
2. Fermentation Culture:
3. A single colony from a verified plate was inoculated into 5 mL of LB medium containing Amp and grown overnight at 37°C with shaking at 220 rpm to serve as the seed culture.
4. The seed culture was transferred at a 1:50 inoculation ratio into 50 mL of M9 minimal medium (supplemented with 0.4% glucose) in a 250 mL conical flask. A negative control was established using BL21(DE3) transformed with the empty pSB1A3 vector.
5. Cultures were incubated at 37°C with shaking at 220 rpm. Samples were taken immediately after inoculation (0 h) and at 1, 3, 8, 12, 24, 36, 48, and 60 hours of cultivation.
6. Sample Analysis:
7. Cell Density (OD600): 1 mL of culture broth was sampled each time, and the OD600 was measured using a spectrophotometer.
8. Violacein Extraction and Quantification:
9. 1 mL of culture broth was centrifuged at 12,000 × g for 2 minutes, and the supernatant was carefully discarded.
10. The cell pellet was thoroughly resuspended in 1 mL of absolute ethanol by vortexing.
11. The suspension was heated in a 65°C water bath for 10 minutes, with vortexing performed 3 times during this period to ensure complete pigment extraction.
12. The sample was centrifuged at 12,000 × g for 5 minutes, and the purple supernatant was collected.
13. The supernatant was appropriately diluted (typically 5-10 fold) with 50% ethanol to ensure its absorbance fell within the linear range of the standard curve.
14. The absorbance of the diluted supernatant was measured at 575 nm, and the violacein concentration was calculated based on the standard curve.
15. Dry Cell Weight (DCW) Estimation: DCW was estimated using a pre-established standard curve correlating OD600 to DCW (under our laboratory conditions, OD600 = 1.0 corresponds to approximately 0.4 g DCW/L).

Results and Analysis:

• Growth Curve: The engineered strain exhibited a slow adaptation phase from 0-8 hours, entered a rapid growth phase from 12-24 hours, and reached the stationary phase around 36 hours, achieving a maximum OD600 of approximately 3.0 (corresponding to ~1.2 g DCW/L). The negative control strain grew faster and reached a higher OD600 in the stationary phase.
• Violacein Production: Violacein synthesis was partially growth-coupled. Production reached approximately 0.15 mg/L at 24 hours, peaked at about 0.24 mg/L at 48 hours, and slightly declined thereafter, potentially due to pigment degradation or cell lysis.
• Specific Yield: The violacein yield per DCW (mg/g DCW) peaked between 24-36 hours (approximately 0.20 mg/g DCW).
• The negative control strain produced no pigment.

Experimental Incident and Reflection:

• Incident: Initial fermentation attempts using basic M9 minimal medium resulted in extremely low violacein yields (<0.05 mg/L) and poor cell growth.
• Solution and Reflection: We hypothesized that the nutritional composition of the M9 medium was insufficient to support the high energy and precursor demands of the heterologous metabolic pathway. Supplementing the M9 medium with 0.5% yeast extract as an additional nutrient source was attempted. This modification led to a significant improvement in both cell growth (maximum OD600 ~3.5) and pigment production (~0.24 mg/L). This experience underscored the critical requirement for a nutrient-rich environment and robust host cell metabolism to achieve high-level production of foreign secondary metabolites.

August 13-20 – Carbon Source Optimization to Enhance Violacein Yield

Experimental Principle:

Although both glycerol and glucose serve as carbon sources, they enter central metabolism via distinct pathways. Glucose is rapidly taken up by the phosphotransferase system (PTS), which can trigger the "glucose effect" (carbon catabolite repression), potentially inhibiting the expression of genes involved in the synthesis of certain secondary metabolites. In contrast, glycerol metabolism does not induce severe carbon catabolite repression. Furthermore, its metabolic pathway—conversion to dihydroxyacetone phosphate before entering glycolysis—may generate a more balanced energy charge and reducing power (NADH/NADPH). This metabolic profile could be more favorable for the synthesis of secondary metabolites like violacein, which requires substantial reducing equivalents.

Experimental Methods:

1. Culture Conditions: Two experimental groups were established:
2. Group 1 (Glucose): M9 + 0.5% Yeast Extract + 0.4% Glucose
3. Group 2 (Glycerol): M9 + 0.5% Yeast Extract + 0.4% Glycerol
4. Fermentation and Analysis: The violacein-producing engineered strain was inoculated into the respective media and cultured at 37°C with shaking at 220 rpm for 24 hours (based on prior results indicating substantial production at this time point). Samples were taken, and OD600 and violacein yield were determined using the methods described previously. Each group was set up with three independent biological replicates (n=3).
5. Statistical Analysis: An unpaired t-test was performed using GraphPad Prism software to compare the violacein yields between the two groups. A p-value of less than 0.05 was considered statistically significant.

Results and Analysis:

• Glucose Group: The average violacein yield was 0.146 ± 0.018 mg/L.
• Glycerol Group: The average violacein yield was 0.281 ± 0.025 mg/L, which is 1.93 times higher than that of the glucose group. The t-test result indicated a p-value < 0.01, demonstrating a highly significant difference.

Experimental Reflection:

This result is highly valuable, as it represents not merely a simple condition optimization but also suggests the importance of the balance between central carbon metabolism and the energy/reducing power demands of heterologous secondary metabolic pathways. Glycerol demonstrated a significant advantage as a carbon source. Future work could involve more systematic fermentation optimization, such as investigating the effects of different carbon-to-nitrogen ratios or using glycerol in combination with other carbon sources. This finding also provides clear direction for enhancing the performance of the reporter module in the subsequent integrated system.

Phase 3: Construction and Validation of the Sterilizing Enzyme System (August 21 – September 5)

August 21-26 – Construction of Chitinase and Glucanase Expression Plasmids

Experimental Principle:

Chitinase hydrolyzes the β-1,4-glycosidic bonds in chitin, a primary structural polymer of N-acetylglucosamine in fungal cell walls. β-1,3-Glucanase hydrolyzes β-1,3-glucans within the cell wall. Their synergistic action can effectively disrupt the integrity of the fungal cell wall, leading to cell lysis and death. The pET-28a(+) vector features a strong, inducible T7/lac promoter for precise regulation by IPTG and an N-terminal 6×His tag. This system enables high-level, inducible expression in E. coli BL21(DE3)—which has the T7 RNA polymerase gene integrated into its genome under the control of the lacUV5 promoter—and facilitates subsequent protein purification via nickel-ion affinity chromatography (Ni-NTA).

Experimental Methods:

1. Gene Synthesis and Cloning: Chitinase (e.g., RmChi44) and β-1,3-glucanase (e.g., MoGluB) genes sourced from different microorganisms were codon-optimized for E. coli, and a sequence encoding a 6×His tag was added to their 5' ends. The optimized gene sequences were synthesized by a commercial gene synthesis company and directly cloned between the NcoI and XhoI restriction sites of the pET-28a(+) vector.
2. Chemical Transformation: The recombinant plasmids, pET28a-Chitinase and pET28a-Glucanase, were separately transformed into E. coli DH5α for plasmid amplification. The plasmids were then extracted and transformed into the expression host, E. coli BL21(DE3). The heat-shock transformation method used was consistent with previous protocols, but selection plates contained kanamycin (50 µg/mL) in LB agar.
3. Positive Clone Verification: Single colonies from the Kan-LB plates were subjected to colony PCR using T7 universal primers and gene-specific primers to verify the size of the inserted fragment. Colonies yielding PCR products of the expected size were sent for Sanger sequencing by a commercial service, using T7 promoter and T7 terminator primers, to confirm that the gene sequences were entirely correct and in the proper reading frame.

Results:

Sequence alignment confirmed that both the chitinase and glucanase genes were correctly inserted into the pET-28a(+) vector, with no base deletions, insertions, or mutations, showing 100% identity with the optimized sequences. The plasmids were successfully constructed.

Experimental Reflection:

Codon optimization is crucial for the efficient expression in E. coli of genes originating from eukaryotes or other prokaryotes with significantly different GC content. It helps prevent translational stalling, inefficiency, or misincorporation of amino acids due to rare codon usage. Outsourcing the synthesis and cloning to a specialized company significantly increased the success rate and efficiency of this step.

August 27-29 – Protein Induction, Expression, and Purification

Experimental Principles:

• IPTG Induction: The T7 promoter in the pET system is regulated by the lac operon. The gene for T7 RNA polymerase in the BL21(DE3) genome is under the control of the lacUV5 promoter. IPTG, an analog of lactose, binds to and inactivates the Lac repressor protein (LacI), thereby relieving its inhibition of the promoter. This activates high-level expression of T7 RNA polymerase, which in turn drives efficient and specific transcription of the target gene.
• Ni-NTA Affinity Purification: The imidazole rings in the polyhistidine (His) tag coordinate with nickel ions (Ni²⁺). Nitrilotriacetic acid (NTA) immobilized on agarose beads chelates Ni²⁺, allowing specific adsorption of His-tagged proteins via coordinate bonds. Washing with buffers containing low concentrations of imidazole removes non-specifically bound impurities. Finally, the target protein bound to Ni²⁺ is competitively eluted using a buffer with a high concentration of imidazole.

Experimental Methods:

1. Small-Scale Test Induction and Expression:

• Single positive colonies of BL21(DE3) harboring pET28a-Chi or pET28a-Glu were picked from plates and inoculated into 5 mL of LB medium containing Kan. Cultures were grown overnight at 37°C.
• Overnight cultures were diluted 1:100 into 50 mL of LB medium with Kan and grown at 37°C with vigorous shaking until OD600 reached approximately 0.6-0.8.
• 1 mL of culture was collected as an uninduced control (0-hour sample). Cells were pelleted by centrifugation and stored at -20°C.
• IPTG was added to the remaining culture to a final concentration of 0.5 mM. Two induction conditions were tested: Group A: induction at 37°C for 4 hours; Group B: induction at 25°C overnight (approximately 16 hours).
• After induction, 1 mL of culture from each condition was collected, and cells were harvested by centrifugation.

2. Solubility Analysis (Preliminary Detection by SDS-PAGE):

• The cell pellets were resuspended in 100 μL of PBS.
• Cells were lysed by sonication on ice (power 300W, pulse 3 seconds, interval 6 seconds, total duration 3 minutes).
• The lysates were centrifuged at 4°C and 12,000 × g for 15 minutes to separate the supernatant (soluble protein) from the pellet (insoluble inclusion bodies).
• The pellet was resuspended in 100 μL of PBS.
• 10 μL samples of both the supernatant and the resuspended pellet were mixed with 5× SDS loading buffer, boiled for 5 minutes, and analyzed by SDS-PAGE (12% separating gel). Gels were stained with Coomassie Blue to assess the form (soluble vs. insoluble) and approximate amount of protein expression.

3. Large-Scale Expression and Purification (Based on Small-Scale Results):

• The 25°C overnight induction condition was selected for scale-up. Overnight seed culture was inoculated 1:100 into 1 L of LB medium with Kan and grown at 37°C to OD600 ≈ 0.6-0.8. IPTG was added to a final concentration of 0.1 mM (further reduced to minimize inclusion body formation), and the culture was transferred to 25°C with shaking at 220 rpm for 16 hours.
• After induction, cells were harvested by centrifugation at 4°C and 6,000 × g for 15 minutes. The cell pellet was resuspended in ice-cold Lysis Buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0).
• Ultrasonic Disruption: Cell lysis was performed on ice using sonication (power 350W, pulse 5 seconds, interval 10 seconds, total duration 15-20 minutes, until the lysate was no longer viscous). This step is critical and must be done on ice to prevent protein degradation due to overheating.
• The lysate was centrifuged at 4°C and 12,000 × g for 30 minutes, and the supernatant (containing soluble protein) was collected.
• Ni-NTA Affinity Purification (using Qiagen Ni-NTA Superflow):
• 1 mL of Ni-NTA resin was packed into a chromatography column and equilibrated with 10 column volumes (CV) of ddH₂O followed by 5 CV of Lysis Buffer.
• The clarified cell lysate supernatant was loaded onto the column slowly at a flow rate of approximately 1 mL/min, and the flow-through was collected.
• The column was washed with 10 CV of Wash Buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH 8.0) to remove non-specifically bound proteins.
• Bound protein was eluted stepwise with 5 CV of Elution Buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0), collecting 1 mL fractions.
• Desalting and Buffer Exchange: The eluted protein was desalted and exchanged into a storage buffer (e.g., PBS or Tris-HCl, pH 7.4) using a PD-10 desalting column (GE Healthcare) or ultrafiltration centrifugal tubes (10 kDa MWCO, Millipore) to remove the high concentration of imidazole.
• Protein Concentration Determination: The concentration of the purified protein was determined using a BCA protein assay kit (Pierce™) according to the manufacturer's instructions, with bovine serum albumin (BSA) as the standard.

Results and Analysis:

• Small-Scale Test: SDS-PAGE analysis revealed that induction at 37°C resulted in the target protein bands (chitinase ~44 kDa, glucanase ~38 kDa) being predominantly present in the pellet (inclusion bodies). In contrast, induction at 25°C significantly enhanced the presence of the target proteins in the supernatant (soluble fraction). Consequently, the strategy of low-temperature (25°C), low-concentration IPTG (0.1 mM) overnight induction was adopted.
• Purification Results: After large-scale purification, SDS-PAGE showed single, sharp predominant bands at the expected molecular weights, with estimated purity exceeding 90%. The BCA assay determined the concentrations to be 1.2 mg/mL for chitinase and 0.9 mg/mL for glucanase.

Experimental Incident and Reflection:

• Major Incident: The initial induction at 37°C with 0.5 mM IPTG resulted in the target protein bands appearing almost exclusively in the pellet (inclusion bodies), with very little in the supernatant, indicating the formation of largely inactive protein aggregates.
• Solution: We promptly consulted the literature and adjusted our strategy, opting for low-temperature induction (25°C) and reduced IPTG concentration (0.1 mM). This significantly slowed the rate of protein synthesis, allowing the cells more time for correct folding and assembly, thereby dramatically increasing the proportion of soluble protein.
• Reflection: High-level expression is not always advantageous, as it can overwhelm the host cell's chaperone systems and proper folding capacity, leading to the formation of inactive inclusion bodies. For heterologous proteins, especially larger enzymes, low-temperature, low-concentration induction is the preferred strategy for obtaining soluble, active protein. This experience provided a profound lesson on the necessity of optimizing expression conditions and the pitfalls of blindly pursuing high yield.

September 1 – SDS-PAGE Verification of Protein Expression

Experimental Principle:

In SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), SDS denatures proteins and confers a uniform negative charge, masking the intrinsic charge differences of the proteins. Consequently, the migration rate of proteins in the electric field is primarily determined by their molecular weight, allowing for the estimation of protein size and assessment of purity.

Experimental Methods:

1. Gel Preparation: A 12% separating gel (lower layer) and a 5% stacking gel (upper layer) were prepared.
2. Separating Gel Formula (10 mL): 30% Acrylamide/Bis solution (4.0 mL), 1.5 M Tris-HCl (pH 8.8) (2.5 mL), 10% SDS (100 μL), 10% Ammonium Persulfate (APS) (50 μL), TEMED (5 μL), topped up to 10 mL with water.
3. Stacking Gel Formula (4 mL): 30% Acrylamide/Bis solution (0.67 mL), 1.0 M Tris-HCl (pH 6.8) (0.5 mL), 10% SDS (40 μL), 10% APS (30 μL), TEMED (5 μL), topped up to 4 mL with water.After pouring the gels, a comb was inserted.
4. Sample Preparation: 10 μL of purified protein sample was mixed with 5 μL of 5× SDS loading buffer. The mixture was denatured by boiling at 100°C for 5 minutes in a heating block.
5. Loading and Electrophoresis: Prestained protein marker and the prepared samples were loaded sequentially into the wells. Electrophoresis was performed in 1× SDS running buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS). An initial voltage of 80V was applied while the samples migrated through the stacking gel. Once the bromophenol blue tracking dye entered the separating gel, the voltage was increased to 120V. Electrophoresis continued for approximately 1 hour, until the dye front reached the bottom of the gel.
6. Staining and Destaining: After electrophoresis, the gel was carefully removed and placed in Coomassie Brilliant Blue R-250 staining solution. Staining was performed on a shaking platform for 45 minutes. The staining solution was then replaced with destaining solution (Methanol:Acetic Acid:Water = 4.5:1:4.5). Destaining was carried out on a shaker, with the destaining solution changed several times until the background was clear and the protein bands were distinctly visible.
7. Imaging: The gel was documented using a gel imaging system.

Results and Analysis:

The Coomassie Blue-stained gel showed single, sharp, and intense predominant bands at the expected molecular weights (chitinase ~44 kDa, glucanase ~38 kDa). The positions of these bands aligned correctly with the prestained protein marker. The absence of significant smearing above the bands or smaller molecular weight bands below indicated high protein purity and no apparent degradation.

Experimental Reflection:

Coomassie Blue staining has limited sensitivity for trace amounts of protein (detection limit ~0.1-1 μg per band). If bands appear faint, more sensitive methods such as silver staining (detection limit ~1-10 ng) or Western Blot for specific verification could be considered. In this instance, due to the effective purification resulting in clear, strong bands, Coomassie Blue staining was sufficient for assessing purity and confirming molecular weight.

September 2-5 – Functional Validation of Enzyme Activity (In Vitro Antifungal Assay)

Experimental Principle:

The purified chitinase and glucanase were co-cultured with the target fungus, using Saccharomyces cerevisiae as a model organism. If the enzymes are active, they will degrade the major components of the fungal cell wall (chitin and glucan), leading to structural disruption, osmotic imbalance, and ultimately resulting in cell lysis or growth inhibition. The antifungal efficacy of the enzymes can be quantitatively assessed by comparing the cell density (OD600) or the number of viable cells (CFU) between the experimental groups and the control groups.

Experimental Methods:

1. Yeast Preparation: Saccharomyces cerevisiae was streaked on a YPD plate for activation. A single colony was picked and inoculated into 5 mL of YPD liquid medium, followed by overnight culture at 30°C with shaking at 200 rpm until the stationary phase was reached. Yeast cells were washed twice with PBS buffer and resuspended to an OD600 of 0.1 (approximately 10^6 CFU/mL).
2. Enzyme Reaction Setup: The following reactions were prepared in 1.5 mL microcentrifuge tubes:
3. Experimental Group 1 (Chitinase): 450 μL yeast suspension + 50 μL purified chitinase (final concentration ~100 μg/mL)
4. Experimental Group 2 (Glucanase): 450 μL yeast suspension + 50 μL purified glucanase (final concentration ~100 μg/mL)
5. Experimental Group 3 (Mixed Enzymes): 450 μL yeast suspension + 25 μL chitinase + 25 μL glucanase
6. Positive Control (Fungicide): 450 μL yeast suspension + 50 μL amphotericin B solution (final concentration 10 μg/mL)
7. Negative Control 1 (Inactivated Enzymes): 450 μL yeast suspension + 50 μL mixed enzymes inactivated by boiling for 10 minutes
8. Negative Control 2 (Buffer Only): 450 μL yeast suspension + 50 μL PBSEach treatment was performed in triplicate.

1. Incubation and Detection:
2. All reaction tubes were incubated at 30°C with shaking at 150 rpm for 4 hours.
3. After incubation, 100 μL of the reaction mixture was appropriately diluted (e.g., 10-fold) with PBS, and the OD600 was measured.
4. Simultaneously, 100 μL of the reaction mixture was serially diluted (10-fold dilutions). 100 μL from appropriate dilution tubes (e.g., 10^-4, 10^-5) was spread onto YPD plates. The plates were incubated inverted at 30°C for 48 hours, after which colony-forming units (CFU) were counted.
5. Data Analysis: The inhibition rate for OD600 and CFU for each treatment group was calculated relative to the "Buffer Only" control group. Inhibition Rate (%) = [1 - (OD600 or CFU of experimental group / OD600 or CFU of control group)] × 100%.

Results and Analysis:

• OD600 Measurement: Compared to the "Buffer Only" control, the inhibition rates were 25% for the chitinase group, 30% for the glucanase group, 55% for the mixed enzyme group, and 95% for the positive control (amphotericin B). The negative control (inactivated enzymes) showed only a 3% inhibition rate, indicating that the antifungal effect was due to the enzymatic activity.
• CFU Counting: The results were consistent with the OD600 trend. The mixed enzyme treatment reduced the viable cell count by approximately one order of magnitude (inhibition rate ~90%), which was significantly stronger than the single enzyme treatments (inhibition rates ~40-50%).

Conclusion:

The purified chitinase and glucanase both possess in vitro antifungal activity. Furthermore, when used in combination, they exhibit a significant synergistic effect, effectively inhibiting the growth of Saccharomyces cerevisiae.

Experimental Reflection:

The in vitro experiment successfully demonstrated the direct antifungal capability of the enzymes under ideal conditions. However, when considering practical application on strawberry surfaces, environmental factors (e.g., temperature, humidity, pH, strawberry surface wax, and native microbiota) may affect enzyme stability and efficacy. A crucial next step is to conduct strawberry fruit inoculation experiments (for instance, inoculating strawberries with Botrytis cinerea and then applying the enzyme solution) to validate the practical preservative effect in a scenario that more closely mimics real-world conditions.

Overall Project Reflection and Outlook

Systems Thinking:

This project entailed a comprehensive practice of the synthetic biology "Design-Build-Test-Learn" (DBTL) cycle. From the screening and characterization of biosensor components (promoters) to the independent construction and optimization of functional modules (the violacein reporter system and the sterilizing enzyme system), and finally to preliminary functional validation, the project honed our capabilities in systems engineering thinking and modular design.

Problem-Solving Skills:

The experiments presented numerous challenges, ranging from PCR condition optimization and fluorescence background interference to protein expression forming inclusion bodies and low fermentation yields. Each instance of troubleshooting served as a valuable learning experience, teaching us how to systematically analyze problems (e.g., identifying whether issues stemmed from reagents, methods, or conditions), formulate reasonable hypotheses, and design controlled experiments for verification. Cultivating this analytical and problem-solving ability is far more significant than merely achieving a successful experimental outcome.

Biosafety and Ethical Considerations:

The engineered strains utilized in this project (DH5α, BL21) are standard laboratory safety strains with no survival advantage in natural environments. All experimental waste containing recombinant DNA was autoclaved prior to disposal. The project's aim is to develop a potential, environmentally friendly biocontrol method to reduce reliance on chemical pesticides, aligning with the ethical imperative of synthetic biology to benefit society.

Limitations:

Currently, each module has been validated independently. The ultimate challenge lies in integrating the VOC sensing module (PgrpE), the violacein reporter module, and the bactericidal module (Chitinase/Glucanase) into a single cell or a co-culture system, and testing its overall performance under simulated or real strawberry storage conditions. This integration may encounter new challenges, such as interference between modules, excessive metabolic burden, and the need for temporal coordination of different functions.

Future Directions:

• System Integration: Construct multi-plasmid systems or utilize gene-editing technologies to integrate different modules into the genome, achieving an integrated "Sense-Report-Act" system.
• Multi-Cellular Systems: Explore the use of co-culture systems where different engineered strains are assigned specific roles (sensing, reporting, and killing), leveraging population cooperation to reduce the metabolic burden on individual cells.
• Practical Application Validation: Conduct small-scale strawberry preservation trials to evaluate the early warning capability and preservative efficacy of the integrated system under real-world conditions.
• Circuit Optimization: Incorporate signal amplification modules (e.g., positive feedback loops) or time-delay switches to create a more sensitive and intelligent system response.
• Chassis Optimization: Consider employing chassis microorganisms better suited for plant surface colonization and more tolerant of environmental stresses, such as certain probiotics or plant endophytes.