iGEM 2025 — Team squirrel-southeast

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
{% block page\_content %}

Content

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.

![A Diagram](https://static.igem.wiki/teams/5879/notebook/img1.webp)

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™).

![A Diagram](https://static.igem.wiki/teams/5879/notebook/img2.webp)

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.

![A Diagram](https://static.igem.wiki/teams/5879/notebook/img3.webp)

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.

![A Diagram](https://static.igem.wiki/teams/5879/notebook/img4.webp)

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).

![A Diagram](https://static.igem.wiki/teams/5879/notebook/img5.webp)

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.

![A Diagram](https://static.igem.wiki/teams/5879/notebook/img6.webp)

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.

![A Diagram](https://static.igem.wiki/teams/5879/notebook/img7.webp)

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.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

Content

* [Summary](#section-1)
  + [Cycle 1: Validation of VOC-Responsive Promoters](#section-2)
    - [Design](#subsection-3)
    - [Build](#subsection-4)
    - [Test](#subsection-5)
    - [Learn](#subsection-6)
  + [Cycle 2: Violacein Expression](#section-7)
    - [Design](#subsection-8)
    - [Build](#subsection-9)
    - [Test](#subsection-10)
    - [Learn](#subsection-11)
  + [Cycle 3: Strawberry Spoilage Detection](#section-12)
    - [Design](#subsection-13)
    - [Build](#subsection-14)
    - [Test](#subsection-15)
    - [Learn](#subsection-16)
  + [Cycle 4: Sterilization System](#section-17)
    - [Design](#subsection-18)
    - [Build](#subsection-19)
    - [Test](#subsection-20)
    - [Learn](#subsection-21)

# Summary

Our team has successfully developed an E. coli-based system for monitoring strawberry spoilage and implementing sterilization, demonstrating the innovative application of synthetic biology in the field of agricultural product preservation. This system was optimized through four rounds of the DBTL (Design-Build-Test-Learn) cycle, ensuring the reliability and practicality of the engineering outcome.

Initially, within the VOC-responsive system module, we constructed reporter gene circuits driven by various promoters. This enables the engineered bacteria to detect characteristic volatile organic compounds (VOCs)—such as 1-octanol, 1-octen-3-ol, and phenylethyl alcohol—released during strawberry spoilage. Detection triggers reporter gene expression, leading to the synthesis of visible violacein, thereby achieving early warning of spoilage. This DBTL iteration focused on optimizing promoter specificity and sensitivity.

Subsequently, in the sterilization and control module, we successfully expressed and purified active preparations of chitinase and glucanase in E. coli. When sprayed onto strawberry surfaces in vitro, these enzyme preparations effectively inhibit the growth of common spoilage fungi, such as Botrytis cinerea, demonstrating a green and safe antifungal effect. This DBTL cycle concentrated on the engineering optimization of enzyme expression and application validation.

The successful construction and validation of these modules not only provide an innovative solution for addressing postharvest strawberry spoilage but also establish a solid foundation for expanding the application of synthetic biology in agricultural product preservation and food safety. Through multiple DBTL iterations, we ensured the engineering success of the system, highlighting a closed-loop optimization process from design to practical application.

![Summary Diagram](https://static.igem.wiki/teams/5879/engineering/img1.webp)

Figure 1 Engineering: Design - Build - Test - Learn

# Cycle 1: Validation of VOC-Responsive Promoters

## Design

This experiment aimed to detect the specificity of responses to Volatile Organic Compounds (VOCs). We designed genetic circuits responsive to VOCs, based on four different promoters, using the plasmid pSB1A3 as the vector. The recA promoter (PrecA) was designated to sense VOCs associated with DNA damage, the grpE promoter (PgrpE) to respond to cytotoxic VOCs, the soxS promoter (PsoxS) to detect VOCs related to oxidative stress, and the lasI promoter (PlasI) to recognize the quorum-sensing signal molecule 3OC12-HSL. All promoters were ligated upstream of the mRFP gene via restriction enzyme digestion and ligation, forming "promoter-mRFP" expression units. Consequently, upon VOC exposure, promoter activation drives mRFP expression, enabling the visual detection of VOCs through changes in fluorescence signal. Our design leverages the specific response mechanisms of these promoters, combined with the fluorescent reporting function of mRFP, to construct an engineered bacterial detection system capable of rapidly sensing multiple types of VOCs.

![Design Diagram](https://static.igem.wiki/teams/5879/engineering/img2.webp)

Figure 2 Construction and Detection of VOCs Response System

## Build

The nucleotide sequences of the PrecA, PgrpE, PsoxS, and PlasI promoters were obtained via gene synthesis. Using the pSB1A3 plasmid as the backbone, the four promoters were individually cloned upstream of the mRFP gene using the Beyotime Seamless Cloning Kit to construct the recombinant plasmids. These recombinant plasmids were then transformed into *E. coli* DH5α and BL21(DE3) competent cells. Positive clones were selected on LB agar plates containing 100 μg/mL ampicillin. Correct construction was confirmed by sequencing (performed by Tsingke), resulting in the final engineered bacterial strains.

![Build Diagram](https://static.igem.wiki/teams/5879/engineering/img3.webp)

Figure 3 Agarose gel electrophoresis verification of PCR results

## Test

### Response of the Four Promoters to VOCs

To evaluate the ability of the engineered strains to recognize various VOCs, the bacteria were first cultured in LB medium at 37°C with shaking. The OD₆₀₀ was monitored every 30 minutes using a FlexStation 3 microplate reader to track growth. Once the culture reached an OD₆₀₀ of 0.6, 200 µL aliquots were transferred to a 96-well plate and co-incubated with a series of VOCs, including gradient dilutions of 1-octanol. After 3 hours of incubation, fluorescence intensity (Excitation 584 nm / Emission 607 nm) and OD₆₀₀ were measured simultaneously. The Fluorescence/OD₆₀₀ ratio was calculated to normalize for differences in cell density.

The results demonstrated that each engineered strain produced a significant fluorescent signal specifically in response to its corresponding VOC. The signal intensity increased with both VOC dosage and incubation time, indicating that the promoters were not readily induced by non-cognate substances. The growth curves of the induced cultures nearly overlapped with those of the blank control groups, confirming that the tested VOCs did not affect bacterial proliferation at the concentrations used. Based on these data, a dose-response heatmap was generated, which visually represents the specific response fingerprint of each promoter across the VOC matrix. This quantification provides a foundational basis for subsequent development of multiplexed detection systems.

![Test Diagram](https://static.igem.wiki/teams/5879/engineering/img4.webp)

Figure 4 Response of different reporter strains to various VOCs

### Comparison of Response Fold-Changes Across Different Promoters to VOCs

The objective was to compare the response intensity of the four VOC-responsive systems, each constructed with a different promoter, in order to identify the most sensitive promoter to the VOCs released during the spoilage process. Through this experiment, we aimed to determine the optimal responsive element, thereby establishing a foundation for constructing the subsequent colorimetric reporter system. This will enable efficient detection and visualization of strawberry spoilage signals.

![Test Diagram](https://static.igem.wiki/teams/5879/engineering/img5.webp)

Figure 5 Characterization of the response characteristics of four promoters to VOCs at the 4-hour time point

(A) Comparison of induction fold changes for VOCs across groups (4h) relative to the control group without VOCs. Error bars represent ± standard deviation (n=3 biological replicates). Statistical significance was determined by one-way ANOVA followed by Tukey's post hoc test. (B) Evaluation of promoter response to VOCs. The number of signals is proportional to the induction fold change.

As the results show, the fluorescence fold-change measured after 4 hours of induction demonstrated that the grpE promoter exhibited the most outstanding performance in detecting the three target VOCs: 1-octanol, 1-octen-3-ol, and phenylethyl alcohol.

* When exposed to 1-octanol, the BL21-grpE-mRFP strain showed a significantly higher Fluorescence/OD₆₀₀ ratio compared to the control (CK) and strains with other promoters. It also achieved the highest response fold-change, indicating the strongest activation effect of grpE by this VOC.
* Under treatment with 1-octen-3-ol, grpE similarly demonstrated the strongest response, with both its fluorescence output and response fold-change significantly leading those of other promoters, confirming its high sensitivity to this VOC.
* For phenylethyl alcohol, while variations existed in the responses among the different promoters, grpE still maintained a high fluorescence level, demonstrating good response stability.

In contrast, the soxS promoter showed a weak response. Its Fluorescence/OD₆₀₀ ratio increased only marginally after treatment with most VOCs, indicating a low association between soxS and these compounds, making effective activation difficult. The *lasI* and *recA* promoters showed some response under specific VOC conditions, but their overall performance levels were markedly lower than that of grpE, indicating limited sensing capability.

In summary, the grpE promoter demonstrated the highest sensitivity and stability in responding to all three target VOCs, identifying it as the most suitable candidate to serve as the core sensing element for constructing the subsequent biosensor.

### Response of the PgrpE Reporter Strain to a Mixed VOC

Based on the previous screening which identified the PgrpE promoter as having the best response intensity to individual VOCs, this experiment aimed to further evaluate the response characteristics of the PgrpE reporter strain to a mixed VOC environment simulating strawberry spoilage (with a phenylethyl alcohol : 1-octanol : 1-octen-3-ol ratio of 6:1:1). By measuring the response fold-change, we sought to validate the sensitivity and applicability of the PgrpE promoter within a complex VOC milieu, thereby providing experimental support for constructing an early detection system for strawberry spoilage.

![Test Diagram](https://static.igem.wiki/teams/5879/engineering/img6.webp)

Figure 6 Response fold of PgrpE reporter strains to the mixed VOCs at different time points. According to the composition distribution of VOCs, the ratio of phenylethyl alcohol:1-octanol:1-octen-3-ol in the VOC mixture is 6:1:1.

The results demonstrated that under treatment with the mixed VOCs, the induction fold-change (FC) of the PgrpE-mRFP reporter strain showed a sustained increasing trend over time (Figure 6). At 2 hours post-treatment initiation, the FC was approximately 1.3-fold, indicating the system's capability to detect VOC signals within a short timeframe. The FC increased to 2.6-fold at 4 hours, reached 3.2-fold at 6 hours, and stabilized at a high level of around 3.5-fold after 10 hours. This demonstrates that PgrpE exhibits a rapid response and a significant signal amplification effect to the mixed VOC signal, further supporting its selection as the superior promoter.

Therefore, based on testing with individual VOCs like 1-octanol and their mixtures, the fluorescence signal mediated by the grpE promoter was significantly stronger than those mediated by lasI*,* recA, and soxS, and it produced a sustained response to the mixture, confirming its status as a superior VOC-responsive promoter.

## Learn

The experimental results confirm that our engineered bacterial system can rapidly and visually detect various Volatile Organic Compounds (VOCs). However, in practical applications, VOC concentrations and combinations are often more complex, and a single promoter may suffer from issues like cross-reactivity or insufficient sensitivity. Therefore, future work could involve collecting comprehensive fluorescence/OD₆₀₀ response matrices to a wider array of VOC stimuli. Integrating these datasets with machine learning algorithms could enable the development of predictive models for the accurate identification of VOC types and concentrations. Simultaneously, model feedback could guide the optimization of promoter combinations and expression systems, continuously enhancing the accuracy, sensitivity, and practicality of the detection system to provide a more reliable biosensing solution for VOC monitoring in complex environments.

# Cycle 2: Violacein Expression

## Design

This experiment aimed to construct an engineered bacterium capable of producing violacein, providing a reliable color-based visual reporting system for the strawberry spoilage biosensor. We used *E. coli* BL21(DE3) as the chassis. We designed a plasmid containing the constitutive strong promoter *PJ23100*, the medium-strength Ribosome Binding Site (RBS) B0034, and the violacein biosynthetic gene cluster (vioAB and vioCDE). The constitutive expression of violacein leads to substantial accumulation of the purple pigment in the engineered bacteria. The effectiveness of the reporting system was confirmed via absorbance readings.

## Build

During the construction process, we first performed codon optimization of the complete violacein biosynthetic gene cluster (*vioABCDE*) for E. coli and completed the full gene synthesis according to BioBrick standards. Subsequently, this optimized fragment was cloned into the high-copy-number plasmid vector pSB1A3 via *EcoRI/XbaI* restriction sites, successfully yielding the recombinant plasmid p1A3-vioABCDE. Correct construction was confirmed by restriction digestion verification and sequencing. This plasmid was transformed into *E. coli* BL21 competent cells, and positive clones were selected using ampicillin resistance. Initial screening was facilitated by the obvious purple colony phenotype. Finally, the verified correct engineered strain was prepared as a glycerol stock and stored at -80°C for future use.

![Build Diagram](https://static.igem.wiki/teams/5879/engineering/img7.webp)

Figure 7 Construction of Violette Plasmid

## Test

We established a reliable method for the quantitative detection of violacein by plotting a standard absorbance curve at a wavelength of 570 nm. The production strain was cultured in M9 minimal medium supplemented with either glucose or glycerol as the carbon source. After a 24-hour incubation period, violacein was extracted separately from both the cell pellet and the supernatant using an ethanol-based extraction method combined with sonication. The yields under different culture conditions were then quantitatively compared. The results demonstrated that detection via absorbance at 570 nm effectively ensured its visualization capability.

![Test Diagram](https://static.igem.wiki/teams/5879/engineering/img8.webp)

Figure 8 Violet compound scan spectrum and standard curve preparation

![Test Diagram](https://static.igem.wiki/teams/5879/engineering/img9.webp)

Figure 9 Detection of violacein content in the produced bacteria

## Learn

The genetic construct successfully synthesized violacein, thereby validating the functional effectiveness of the circuit. Notably, supplementing the culture medium with glycerol significantly enhanced violacein production compared to glucose, demonstrating superior performance and indicating that carbon source optimization is crucial for yield maximization. This engineered strain not only provides a functional visual reporting system but also establishes an optimized platform for product synthesis. Subsequent work will focus on replacing the constitutive promoter with a promoter activated by strawberry VOCs and, under glycerol-supplemented conditions, further enhancing the response amplitude of the sensor.

# Cycle 3: Strawberry Spoilage Detection

## Design

This project aims to develop a biosensor for the rapid, sensitive, and visual detection of early-stage strawberry spoilage. We selected *E. coli* DH5α (Beyotime, D0351) as the chassis organism and constructed a genetic circuit comprising a spoilage-related Volatile Organic Compound (VOC) response promoter (PgrpE) and the violacein biosynthetic operon (*vioABCDE*). The target VOC mixture simulating spoilage consists of phenylethyl alcohol, 1-octanol, and 1-octen-3-ol in a 6:1:1 ratio. When the engineered bacteria sense the characteristic VOCs released during strawberry spoilage, the PgrpE promoter is activated, driving the expression of the downstream violacein operon. This results in a visible purple color change, reporting the spoilage signal and enabling rapid, on-site detection without the need for complex instruments.

## Build

During the construction phase, we first successfully amplified the optimally screened VOC-responsive promoter *PgrpE* and all five genes required for violacein biosynthesis (vioABCDE) using PCR. Subsequently, employing molecular cloning techniques such as restriction digestion and ligation, we sequentially linked PgrpE with the violacein operon and inserted the assembly into the standard vector pSB1C3, yielding the recombinant plasmid pSB1C3-PgrpE-vioABCDE. This created the complete "sense-and-report" genetic circuit. The correctness of the recombinant plasmid was verified through colony PCR, double restriction enzyme digestion, and sequencing. The verified plasmid was then introduced into E. coli BL21(DE3) via heat shock transformation. Positive clones were selected using antibiotic resistance and prepared as glycerol stocks stored at -80°C. During expression condition optimization, it was discovered that replacing 0.4% glucose with 0.4% glycerol as the carbon source increased violacein production by approximately 1.9-fold. Therefore, M9 medium supplemented with glycerol was established as the optimal working medium for the sensor to enhance the output signal intensity.

![Build Diagram](https://static.igem.wiki/teams/5879/engineering/img10.webp)

Figure 10 Construction of PgrpE-volacein engineered bacteria

## Test

Our team designed a systematic testing protocol: First, strawberry samples with varying degrees of spoilage (fresh, slight, moderate, severe) were prepared either by artificial inoculation with *Botrytis cinerea* or through natural spoilage. Cultures of the engineered strain, grown to the logarithmic phase, were co-incubated with the individual samples in a sealed container for 4-12 hours. A negative control was established using bacterial culture not exposed to any sample, and a positive control used culture exposed to a high concentration of a single VOC.

For signal readout, a qualitative assessment was performed by visually observing whether the bacterial culture developed a purple color. For quantitative analysis (planned), violacein will be extracted using ethanol and its absorbance measured at 575 nm. This data will be used to plot a relationship curve between the signal response and the degree of spoilage, thereby evaluating the sensor's detection range, sensitivity, and quantitative potential. Additionally, the sensor's specificity for strawberry spoilage VOCs will be validated by testing its response to volatile compounds from other common fruits and vegetables.

![Test Diagram](https://static.igem.wiki/teams/5879/engineering/img11.webp)

Figure 11 Strawberry Spoilage Detector Response Flowchart

## Learn

Based on preliminary experimental results, this sensor is expected to effectively respond to strawberry spoilage markers. However, the current system has several limitations, and future improvements will focus on the following areas:

* Performance Enhancement: To improve response sensitivity and kinetic characteristics, we plan to employ directed evolution of the *PgrpE* promoter and introduce signal amplification circuits to shorten the detection time and lower the detection limit.
* Signal Readout: We will develop a portable colorimetric device or a smartphone image analysis algorithm to achieve quantification and standardization of the output signal, overcoming the subjectivity of visual interpretation.
* Application Robustness: Efforts will be made to enhance the environmental robustness of the engineered strain. We intend to use metabolic engineering or microencapsulation techniques to improve the stability of E. coli DH5α under real-world conditions.
* Specificity: Through transcriptomic analysis and the design of logic gate circuits capable of responding to two or more characteristic VOCs simultaneously, we aim to further enhance system specificity and reduce cross-reactivity.

Looking forward, we will advance the integration of the sensor into portable detection devices and explore its coupling with a bactericidal module to create a closed-loop "sense-report-act" system. Furthermore, leveraging the platform's extensibility, the sensing module can be readily replaced to quickly adapt the system for spoilage monitoring of other perishable fruits and vegetables, thereby broadening its application scenarios.

# Cycle 4: Sterilization System

## Design

While our detector successfully identifies strawberry spoilage, interviews with stakeholders (HP) revealed that detection alone is not a complete solution. To further reduce losses, we proposed a proactive method: spraying chitinase and glucanase onto strawberries before transport. These enzymes target and degrade the core structural polymers (chitin and glucan) of the fungal cell wall at the molecular level. This disruption causes structural collapse and functional loss of the cell wall, ultimately leading to fungal inhibition or death, thereby reducing strawberry spoilage caused by fungi.

## Build

* The optimized *chiA* gene was amplified by PCR, digested with *NdeI* and *XhoI* restriction enzymes, and inserted into the corresponding site of the pET-28a(+) plasmid. This vector drives target gene expression via the T7/lac promoter, incorporates an N-terminal 6×His tag to facilitate subsequent protein purification and detection, and carries a kanamycin resistance (Kan^R) marker.
* Similarly, the optimized *gluA* gene was amplified by PCR, digested with *BamHI* and *NotI* restriction enzymes, and cloned into the corresponding site of the pCDFDuet-1 plasmid. This vector also regulates expression with a T7/lac promoter and carries a spectinomycin resistance (Spec^R) marker.
* Following digestion, the target gene fragments and plasmid backbones were ligated to construct the recombinant plasmids, which were then transformed into *E. coli* DH5α as the cloning host. The resulting clones were verified by double restriction enzyme digestion. Positive clones were selected for plasmid extraction and sequencing to confirm the correctness and integrity of the inserted fragments.

![Build Diagram](https://static.igem.wiki/teams/5879/engineering/img12.webp)

Figure 12 Construction of pET28a-Chitinase and pET28a-Glucanase vectors

## Test

### Validation of Chitinase and Glucanase Expression

To confirm the successful expression of the target proteins, our team conducted *induced cultivation* of the recombinant strains and performed qualitative protein analysis using SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). Bacterial cells were harvested under the respective induction conditions, lysed, and soluble proteins were extracted for electrophoretic separation on a gel.

The results showed that specific protein bands corresponding to the theoretical molecular weights were observed in the experimental samples: PelB-ChiA exhibited a band at approximately 70 kDa, and PelB-GluB showed a band at around 65 kDa, both matching the expected sizes. No corresponding bands were detected in the empty vector control group, confirming that these bands originated from the expression of the target genes.

![Test Diagram](https://static.igem.wiki/teams/5879/engineering/img13.webp)

Figure 13 Verification of Chitinase and Glucanase Expression

### Functional Validation of Chitinase and Glucanase Expression

This experiment aimed to validate the in vitro broad-spectrum antifungal activity of the purified Chitinase and β-1,3-Glucanase. Using the model fungus—*Saccharomyces cerevisiae*—as the target organism, we assessed the inhibitory effects on fungal growth for the individual enzymes and their combination. Since chitin and β-1,3-glucan are core structural components of the cell walls in the vast majority of fungi—including those causing strawberry spoilage such as *Botrytis cinerea*, *Rhizopus spp.*, and *Penicillium spp*.—this experiment provides crucial functional evidence to support the subsequent application of these two enzymes on strawberry surfaces for the broad-spectrum control of various fungi responsible for postharvest spoilage.

![Test Diagram](https://static.igem.wiki/teams/5879/engineering/img14.webp)

Figure 14 Synergistic inhibition of *S. cerevisiae* by a combination of chitinase and glucanase.

Table 1. Fungistatic activity of chitinase and glucanase against *S. cerevisiae*.

![Test Diagram](https://static.igem.wiki/teams/5879/engineering/img15.webp)

(Note: CFU: Colony Forming Unit. The relative survival rate was calculated based on the viable count of Group 2. Statistical significance was determined by one-way ANOVA; *p* < 0.001 compared to Group 2.)

The quantitative results from the antifungal assay clearly characterized the inhibitory effects of the two enzymes individually and in combination (Figure 14, Table 1). Data from colony counting revealed:

* Negative Control (Group 2, Heat-inactivated enzymes): The yeast colony count was 7×10⁷ CFU/mL, set as the baseline survival rate (100%). This confirmed that non-active components in the enzyme preparation did not affect yeast growth.
* Chitinase Alone (Group 3): Reduced the viable yeast count to 1×10⁷ CFU/mL, corresponding to a relative survival rate of 14.3%. This demonstrates that chitinase alone can effectively degrade fungal cell walls and inhibit cell survival.
* Glucanase Alone (Group 4): Showed stronger antifungal activity, reducing the yeast colony count to 3×10⁶ CFU/mL, with a relative survival rate of only 4.3%.
* Enzyme Combination (Group 5): The most significant effect was observed with the dual-enzyme combination, which drastically reduced the viable yeast count to 1.4×10⁶ CFU/mL, yielding a very low relative survival rate of 2.0%. This result was significantly superior to either single-enzyme treatment group (*p* < 0.001), revealing a powerful synergistic antifungal effect between the two enzymes.

## Learn

Through this experimental cycle, we successfully achieved high-yield expression in *E. coli* and obtained biologically active *Chitinase* and *β-1,3-Glucanase*. Functional validation using *Saccharomyces cerevisiae* as a model fungus demonstrated that the two enzymes, working *synergistically*, significantly reduced cell viability, confirming their excellent activity and application potential in degrading fungal cell walls.

These results provide crucial support for our subsequent application research. In the future, we plan to further evaluate the performance of the dual-enzyme system in practical strawberry preservation. This will involve testing its efficacy in inhibiting fungal infection through methods like spraying or coating on fruit surfaces. Concurrently, we will explore optimizations for enzyme stability and adjustments to the formulation ratio to achieve more durable and efficient protective effects. Through these refinements, we aim to integrate this module with our previously developed VOC detection system, ultimately constructing an intelligent, closed-loop "detection-response-inhibition" bioprotection system.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

Content

* [I. Construction of Recombinant Strains](#section-1)
  + [1. Plasmid Construction](#subsection-2)
  + [2. Transformation and Verification](#subsection-3)
* [II. Screening of VOCs-Responsive Promoters](#section-4)
  + [1. Bacterial Culture and VOCs Induction](#subsection-5)
  + [2. Fluorescence and Optical Density Measurements](#subsection-6)
* [III. Violacein Production and Quantification](#section-7)
  + [1. Fermentation and Sampling](#subsection-8)
  + [2. Extraction and Quantification of Violacein](#subsection-9)
* [IV. Expression, Purification, and Functional Assay of Antifungal Enzymes](#section-10)
  + [1. Protein Expression and Purification](#subsection-11)
  + [2. In Vitro Antifungal Activity Assay](#subsection-12)
* [V. General Molecular Biology Procedures](#section-13)
  + [1. PCR Amplification (Conventional or High-Fidelity)](#subsection-14)
  + [2. Colony PCR](#subsection-15)
  + [3. Agarose Gel Electrophoresis and Gel Extraction](#subsection-16)
  + [4. Double Digestion and Ligation](#subsection-17)
  + [5. Plasmid Miniprep / Midiprep](#subsection-18)

# I. Construction of Recombinant Strains

## 1. Plasmid Construction

### VOCs-Responsive Reporter Plasmids:

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*.

### Violacein Production Plasmid:

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).

### Antifungal Enzyme Expression Plasmids:

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.

## 2. Transformation and Verification

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.

# II. Screening of VOCs-Responsive Promoters

## 1. Bacterial Culture and VOCs Induction

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.

## 2. Fluorescence and Optical Density Measurements

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.

# III. Violacein Production and Quantification

## 1. Fermentation and Sampling

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).

## 2. Extraction and Quantification of Violacein

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.

# IV. Expression, Purification, and Functional Assay of Antifungal Enzymes

## 1. Protein Expression and Purification

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.

## 2. In Vitro Antifungal Activity Assay

*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) Negative control: 100 μL yeast suspension + 100 μL heat-inactivated (95 °C, 15 min) enzyme mixture (each enzyme = 0.5 mg/mL).

(2) Chitinase: 100 μL yeast + 100 μL chitinase (0.5 mg/mL).

(3) Glucanase: 100 μL yeast + 100 μL glucanase (0.5 mg/mL).

(4) Enzyme combination: 100 μL yeast + 50 μL chitinase + 50 μL glucanase (each = 0.25 mg/mL).

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.

# V. General Molecular Biology Procedures

## 1. PCR Amplification (Conventional or High-Fidelity)

Reaction mixture (25 μL):

* 10× PCR buffer: 2.5 μL
* MgCl₂ (if not included): 0.75 μL (25 mM)
* dNTPs (10 mM each): 0.5 μL
* Forward primer (10 μM): 0.5 μL
* Reverse primer (10 μM): 0.5 μL
* Template DNA: 1 μL (1–100 ng plasmid or 50–200 ng genomic DNA)
* DNA polymerase (Taq or high-fidelity, e.g., Phusion/Q5): 0.25–0.5 μL
* Sterile ddH₂O to 25 μL total.

Cycling conditions:

* Initial denaturation: 95 °C, 2–3 min
* 25–35 cycles of: 95 °C 30 s (denaturation) Annealing at Tm − 3 to − 5 °C for 30 s 72 °C extension (30 s/kb for Taq; 15–30 s/kb for high-fidelity)
* Final extension: 72 °C 5–10 min
* Hold at 4 °C.

Notes:

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.

## 2. Colony PCR

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.

## 3. Agarose Gel Electrophoresis and Gel Extraction

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.

## 4. Double Digestion and Ligation

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.

## 5. Plasmid Miniprep / Midiprep

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.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}

Content

* + [I. Screening of VOC-Responsive Promoters](#section-2)
    - [Experimental Objective](#subsection-3)
    - [Experimental Methods](#subsection-4)
    - [Experimental Results](#subsection-5)
  + [II. Construction and Optimization of the Violacein Production System](#section-6)
    - [2.1 Establishment of the Absorption Spectrum and Standard Curve for Violacein](#subsection-7)
    - [2.2 Analysis of Violacein Production in Engineered Bacteria](#subsection-8)
    - [2.3 Glycerol Enhancement of Violacein Production](#subsection-9)
    - [2.4 Analysis of VOC Response by PgrpE-VioABCDE Reporter Strain](#subsection-10)
  + [III. Construction and Validation of the Antifungal System](#section-11)
    - [3.1 Expression Validation of Chitinase and Glucanase](#subsection-12)
    - [3.2 Functional Validation of Chitinase and Glucanase Expression](#subsection-13)

This study focuses on the early identification and prevention of postharvest decay in strawberries, constructing a comprehensive synthetic biology system comprising three components: detection, reporting, and response. First, by screening VOC-responsive promoters, we established a biosensing module capable of recognizing volatile organic compounds (VOCs) released during the decay process, enabling sensitive capture of spoilage signals. Second, we integrated the highly responsive promoter *PgrpE* with the violacein chromogenic pathway (*vioABCDE*) to develop a detection system that converts invisible spoilage gas signals into visible color changes. Finally, we constructed a synergistic antimicrobial module combining *Chitinase* and *β-1,3-Glucanase* to achieve active defense against fungal infection. Through step-by-step validation of these three experimental modules, we realized a closed-loop design spanning from signal perception and colorimetric reporting to active prevention, providing a biological foundation and technical support for the intelligent monitoring and preservation of strawberries and other perishable fruits.

![A Diagram](https://static.igem.wiki/teams/5879/results/img1.webp)

Figure 1. Integrated Synthetic Biology System for Strawberry Spoilage Detection and Inhibition

# I. Screening of VOC-Responsive Promoters

## Experimental Objective

This experiment aims to screen for VOC-responsive promoters that can be used to detect early spoilage signals in postharvest perishable fruits such as strawberries.

Strawberries are susceptible to fungal infections (e.g., *Botrytis cinerea*) during harvesting, transportation, and storage. During growth and metabolism, fungi release characteristic volatile organic compounds (VOCs), such as 1-octanol, 1-octen-3-ol, and phenylethyl alcohol. These VOCs can serve as early molecular indicators of fruit spoilage. By evaluating the activation of four VOC-responsive promoters (*PsoxS*, *PlasI*, *PrecA*, *PgrpE*) in response to these three VOCs, using red fluorescent protein (RFP) as a reporter gene for visual and quantitative analysis, we aim to identify the promoter with the strongest response to spoilage-related VOCs and the lowest background noise. This will provide a foundation for constructing a VOC detection and fruit spoilage early-warning system.

## Experimental Methods

### Genetic Engineering Construction

Four VOC-responsive promoters (*PsoxS*, *PlasI*, *PrecA*, *PgrpE*) were synthesized and cloned into the standard iGEM plasmid pSB1A3 vector using seamless cloning technology. These promoters were placed upstream of the reporter gene *mRFP* to construct a "promoter-RFP" expression detection system. *Escherichia coli* DH5α (Beyotime, D0351) was used for plasmid amplification, and positive clones were selected using ampicillin (100 μg/mL, Amp) resistance screening. Single colonies were picked for plasmid extraction and sent for sequencing verification (Tsingke, Beijing). After confirming the correct construction, the plasmids were transformed into the expression strain BL21(DE3) (Beyotime, D1009S) for subsequent VOC response experiments. The final engineered strains were stored at -80°C with 25% (v/v) glycerol as a cryoprotectant.

![Experimental Methods Diagram](https://static.igem.wiki/teams/5879/results/img2.webp)

Figure 2. Plasmid Construction and Transformation Verification

### Bacterial Culture and VOC Induction

Verified engineered strains were inoculated into LB liquid medium (10 mL, in 50 mL conical flasks) containing ampicillin and cultured at 37°C with shaking at 250 rpm. The OD₆₀₀ was monitored in real-time to ensure the cells were in the logarithmic growth phase. When the OD₆₀₀ reached 0.6, the bacterial culture was aliquoted into a 96-well plate, with 90 μL per well as the detection system. VOC stock solutions (1-octanol, 1-octen-3-ol, phenylethyl alcohol) were prepared as 0.1% (v/v) aqueous solutions and then diluted 10³-fold to the experimental concentration. In the experiment, 10 μL of different VOC solutions were added to each well, ensuring the final system simulated the actual VOC concentrations during strawberry spoilage. A control group without VOCs was included, and the total experimental system volume was 100 μL. The 96-well plate was incubated statically at 37°C for 4 hours to allow full diffusion of VOCs and interaction with the engineered bacteria, activating the promoters and inducing RFP expression.

### Fluorescence Measurement and Promoter Screening

After incubation, two indicators were measured using a microplate reader:

* Fluorescence Intensity: Red fluorescence of RFP (excitation wavelength 584 nm, emission wavelength 607 nm) was detected to reflect the degree of promoter activation.
* OD₆₀₀ Value: Cell density was measured simultaneously to correct for growth differences between wells.The standardized fluorescence ratio (Fluorescence/OD₆₀₀) was calculated to eliminate interference from variations in bacterial cell numbers, providing accurate data on promoter response strength. The three VOC treatment groups were compared with the control group, and normalized fluorescence histograms were plotted to analyze the response patterns of each promoter under different VOC stimuli. The promoter with the highest sensitivity to the target VOCs and the lowest background signal was selected as the core component for the subsequent VOC detection system, enabling early detection and warning of VOCs during fruit spoilage.

![Experimental Methods Diagram](https://static.igem.wiki/teams/5879/results/img3.webp)

Figure 3. Screening Process for VOC-Responsive Promoters

## Experimental Results

### 1. PCR and Agarose Gel Verification

We first performed PCR validation on the reporter gene *mRFP* and the four recombinant plasmids containing different promoters. Target fragments were amplified using specific primers, and the PCR products were analyzed by 1.2% agarose gel electrophoresis, revealing clear single bands (Figure 1). The results showed that the *mRFP* fragment size matched the expected size (approximately 678 bp), while the amplified fragments of the four "promoter + *mRFP*" constructs also corresponded to their theoretical sizes: *PrecA-mRFP* (~802 bp), *PgrpE-mRFP* (~776 bp), *PsoxS-mRFP* (~740 bp), and *PlasI-mRFP* (~835 bp). All bands were at the correct positions with no significant non-specific amplification. To further confirm the accuracy of the constructs, positive clones were subjected to Sanger sequencing, which confirmed that the sequences were fully consistent with the design, with no frameshift or premature termination mutations detected. In conclusion, all four target plasmids were successfully constructed and are suitable for subsequent functional validation experiments.

![Experimental Results Diagram](https://static.igem.wiki/teams/5879/results/img4.webp)

Figure 4. Verification of PCR results by agarose gel electrophoresis

### 2. Response of *PlasI* to VOCs

![Experimental Results Diagram](https://static.igem.wiki/teams/5879/results/img5.webp)

Figure 5. Construction and Validation of *PlasI+mRFP*

Experimental results showed that the *PlasI* reporter strain exhibited differentiated response patterns after 4-hour exposure to different VOCs (Figure 4). The control group (CK) demonstrated the lowest normalized fluorescence value, representing the baseline expression level of the *PlasI* reporter strain in the non-responsive state.

Under the treatment of 1-octanol and phenylethyl alcohol, the normalized fluorescence value of *PlasI* significantly increased, approximately 2-fold higher than CK, indicating that *PlasI* could be markedly induced by 1-octanol and phenylethyl alcohol. The fluorescence value of the 1-octen-3-ol treatment group was about 1.5 times higher than CK, showing a moderate response. Comprehensive comparison revealed that the sensitivity of *PlasI* to the three VOCs followed the order: 1-octanol > phenylethyl alcohol > 1-octen-3-ol, with 1-octanol and phenylethyl alcohol being the most effective inducer molecules.

![Experimental Results Diagram](https://static.igem.wiki/teams/5879/results/img6.webp)

Figure 6. Response of the PlasI reporter strain to different VOCs (4h)

### 3. Response of PrecA to VOCs

![Experimental Results Diagram](https://static.igem.wiki/teams/5879/results/img7.webp)

Figure 7. Construction and Validation of PrecA+mRFP

Experimental results demonstrated that the *PrecA* reporter strain exhibited differentiated response patterns after 4-hour exposure to different VOCs (Figure 2). The control group (CK) showed the lowest normalized fluorescence value, representing the baseline expression level of the *PrecA* reporter strain in the non-responsive state.

Among the three VOC treatment groups, the *PrecA* reporter strain displayed comparable response intensities under 1-octanol and phenylethyl alcohol treatments, both approximately 1.5-fold higher than CK, indicating that the *PrecA* response system could be significantly induced by these two VOCs. The response intensity of the 1-octen-3-ol treatment group was slightly lower than the other two groups, showing a moderate response. Comprehensive comparison revealed that the sensitivity of *PrecA* to the three VOCs followed the order: 1-octanol > phenylethyl alcohol > 1-octen-3-ol, with 1-octanol being the most effective inducer molecule.

![Experimental Results Diagram](https://static.igem.wiki/teams/5879/results/img8.webp)

Figure 8. Response of the PrecA reporter strain to different VOCs (4h)

### 4. Response of PgrpE to VOCs

![Experimental Results Diagram](https://static.igem.wiki/teams/5879/results/img9.webp)

Figure 9. Construction and Validation of PgrpE+mRFP

Experimental results showed that the *PgrpE* reporter strain exhibited differentiated response patterns after 4-hour exposure to different VOCs (Figure 2). The control group (CK) demonstrated the lowest normalized fluorescence value, representing the baseline expression level of the *PgrpE* reporter strain in the non-responsive state.

Under the treatment of 1-octanol and phenylethyl alcohol, the normalized fluorescence value of the *PgrpE* promoter significantly increased, approximately 2.3-fold higher than CK, indicating that *PgrpE* could be markedly induced by 1-octanol and phenylethyl alcohol. The fluorescence value of the 1-octen-3-ol treatment group was slightly lower than the other treatment groups, but still exhibited relatively strong response intensity. Comprehensive comparison revealed that the sensitivity of *PgrpE* to the three VOCs followed the order: 1-octanol > phenylethyl alcohol > 1-octen-3-ol, with 1-octanol and phenylethyl alcohol being the most effective inducer molecules.

![Experimental Results Diagram](https://static.igem.wiki/teams/5879/results/img10.webp)

Figure 10. Response of the PgrpE reporter strain to different VOCs (4h)

### 5. Response of PsoxS to VOCs

![Experimental Results Diagram](https://static.igem.wiki/teams/5879/results/img11.webp)

Figure 11. Construction and Validation of PsoxS+mRFP

Experimental results demonstrated that the *PsoxS* reporter strain exhibited differentiated response patterns after 4-hour exposure to different VOCs (Figure 2). The control group (CK) showed the lowest normalized fluorescence value, representing the baseline expression level of the *PsoxS* reporter strain in the non-responsive state.

Under 1-octanol treatment, the normalized fluorescence value of the *PsoxS* reporter significantly increased, approximately 3-fold higher than CK, indicating that *PsoxS* could be strongly induced by 1-octanol. The fluorescence values of the 1-octen-3-ol and phenylethyl alcohol treatment groups were also higher than CK, with comparable levels between them, showing an increase of approximately 1.2-fold compared to CK. This suggests that *PsoxS* could also respond to these two VOCs, though the response intensity was significantly lower than that to 1-octanol. Comprehensive comparison revealed that the sensitivity of *PsoxS* to the three VOCs followed the order: 1-octanol > phenylethyl alcohol ≈ 1-octen-3-ol, with 1-octanol being the most effective inducer molecule.

![Experimental Results Diagram](https://static.igem.wiki/teams/5879/results/img12.webp)

Figure 12. Response of the PsoxS reporter strain to different VOCs (4h)

### 6. Comparison of Response Fold Changes of Different Promoters to VOCs

#### Objective

To compare the response intensities of VOC-responsive systems constructed with four different promoters and screen for the promoter most sensitive to VOC signals released during spoilage processes. Through this experiment, we aim to identify the optimal responsive element, providing a foundation for subsequent construction of chromogenic reporter systems, thereby enabling efficient detection and visualization of strawberry spoilage signals.

#### Methods

Correctly constructed VOC-responsive strains with four different promoters were inoculated into LB liquid medium containing ampicillin (10 mL, in 50 mL conical flasks) and cultured in a shaker at 37°C and 250 rpm. The OD₆₀₀ value was monitored in real-time to ensure cells were in the logarithmic growth phase. When OD₆₀₀ reached 0.6, the bacterial suspension was aliquoted into a 96-well plate, with 90 μL per well as the detection system. VOC stock solutions (1-octanol, 1-octen-3-ol, phenylethyl alcohol) were first prepared as 0.1% (v/v) aqueous solutions, then diluted 10³-fold to the required experimental concentrations. During the experiment, 10 μL of different VOC solutions were added to each well, enabling the final system to simulate the actual VOC concentrations during strawberry spoilage. A control group without VOCs was established, with a total system volume of 100 μL. The 96-well plate was statically incubated at 37°C for 4 hours to allow sufficient VOC diffusion and interaction with the engineered bacteria, activating the promoters and inducing RFP expression.

After incubation, the 96-well plate was placed in a microplate reader to detect two parameters:

* Fluorescence intensity: Detection of RFP red fluorescence (excitation wavelength 584 nm, emission wavelength 607 nm), reflecting the degree of promoter activation
* OD₆₀₀ value: Simultaneous measurement of cell density for correcting growth differences between wells

The RFP reporter systems driven by four different promoters were incubated for 4 hours under three VOC conditions simulating strawberry spoilage (1-octanol, 1-octen-3-ol, phenylethyl alcohol), followed by RFP fluorescence measurement with OD₆₀₀ correction. Results were expressed as normalized fluorescence values (Fluorescence/OD) and plotted in Figure 2-6.

Calculation of Normalized Fluorescence Value:

![Experimental Results Diagram](https://static.igem.wiki/teams/5879/results/img13.webp)

To compare the response intensities of four promoters (PsoxS, PlasI, PrecA, PgrpE) to three VOCs (1-octanol, 1-octen-3-ol, phenylethyl alcohol), we calculated response fold changes at the 4-hour incubation time point. This is a commonly used quantitative indicator for evaluating the relative degree of promoter activation by VOCs. Response fold change can reflect promoter sensitivity and specificity - higher fold changes indicate stronger promoter response to specific VOCs.

Response Fold Change Calculation Formula:

For each promoter and each VOC:

Response Fold Change = (Normalized fluorescence value of VOC treatment group) / (Normalized fluorescence value of no-VOC control group)

Evaluation Criteria for Promoter Response to VOCs:

The response intensities of the four promoters to VOCs were ranked and visually represented using corresponding numbers of "+" symbols. The top-ranked promoter is indicated by "++++", while the lowest-ranked promoter is indicated by "+", with others following this pattern.

#### Experimental Results:

As shown in the results, fluorescence fold change measurements after 4 hours revealed that the grpE promoter performed most prominently when detecting the three target VOCs (1-octanol, 1-octen-3-ol, and phenylethyl alcohol). When exposed to 1-octanol, the "fluorescence value/OD₆₀₀" of BL21-grpE-mRFP strain was significantly higher than the control group (CK) and other promoter strains, with the highest response fold change, indicating that grpE had the strongest activation effect for this VOC. Under 1-octen-3-ol treatment, grpE similarly showed the strongest response, with both fluorescence output values and response fold changes significantly leading other promoters, demonstrating grpE's high sensitivity to this VOC. For phenylethyl alcohol, although there were some differences in responses among promoters, grpE still maintained high fluorescence levels, exhibiting good response stability. In contrast, the soxS promoter showed weaker responses. After most VOC treatments, its "fluorescence value/OD₆₀₀" showed limited improvement, indicating low correlation between soxS and these VOCs, making effective response difficult. lasI and recA promoters showed some response under certain VOC conditions, but their overall levels were significantly lower than grpE, indicating limited sensing capabilities. In summary, the grpE promoter demonstrated the highest sensitivity and stability in responses to all three target VOCs, making it the most suitable candidate promoter as the core element for subsequent biosensor construction.

![Experimental Results Diagram](https://static.igem.wiki/teams/5879/results/img14.webp)

Figure 12 Characterization of Response Properties of Four Promoters to VOCs at 4-hour Time Point

(A) Comparison of induction fold changes for each group relative to no-VOC control group (4h). Error bars represent ± standard deviation (n=3 biological replicates). Statistical significance was determined by one-way ANOVA and Tukey's post-hoc test；(B) Evaluation of promoter responses to VOCs. The number of "+" symbols is proportional to induction fold change

#### Discussion:

From the experimental results, it is evident that the four VOC-responsive promoters exhibited significant differences in their responses to different target volatile organic compounds. Among them, the grpE promoter demonstrated the highest fluorescence output and response fold change under all three VOC treatment conditions (1-octanol, 1-octen-3-ol, and phenylethyl alcohol), indicating its highly sensitive and stable response characteristics to multiple VOCs. This finding aligns with the biological function of the grpE promoter in bacterial stress response. grpE typically participates in protein folding and stress regulation processes, enabling rapid initiation of downstream gene expression when exposed to external chemical stimuli, which may explain its strong induction under various VOC stimulations.

In contrast, the soxS, lasI, and recA promoters showed relatively lower response capabilities, suggesting that their regulatory mechanisms may not be directly associated with VOC signals, or that their sensing pathways have limited activity in the *E. coli* BL21 background. Particularly for soxS, its limited response enhancement implies that it is more inclined to sense oxidative stress signals rather than volatile organic compounds.

Comprehensive analysis indicates that the grpE promoter combines the advantages of high response sensitivity, stable signal output, and low background noise, making it the most suitable candidate as the core sensing element for subsequent VOC detection systems. These results provide a solid foundation for the construction of chromogenic modules in this project and validate the feasibility of optimizing detection sensitivity through promoter screening. Future work could further investigate the dynamic response characteristics of grpE across different VOC concentration gradients and explore the regulatory mechanisms at the cellular level to provide additional basis for system optimization.

### 7. Response of the PgrpE Reporter Strain to Mixed VOCs

#### Objective

Based on previous screening that identified the PgrpE promoter as having the strongest response to individual VOCs, this experiment aimed to further evaluate the response characteristics of the PgrpE reporter strain to a VOC mixture simulating the strawberry spoilage environment (phenylethyl alcohol:1-octanol:1-octen-3-ol = 6:1:1). By measuring response fold changes, we validated the sensitivity and applicability of the PgrpE promoter in complex VOC environments, providing experimental evidence for constructing an early strawberry spoilage detection system.

#### Methods

Strain and Culture

The experiment used *Escherichia coli* DH5α carrying the PgrpE promoter-RFP reporter system (construction method as previously described). The strain was inoculated into 10 mL of LB liquid medium containing 100 μg/mL ampicillin (in 50 mL conical flasks) and cultured at 37°C with 250 rpm shaking. OD₆₀₀ was monitored, and subsequent experiments were conducted when the bacterial culture reached mid-logarithmic growth phase (OD₆₀₀ ≈ 0.6).

VOC Mixture Preparation

According to the typical composition ratio of VOCs during strawberry spoilage, a mixed VOC solution was prepared with phenylethyl alcohol:1-octanol:1-octen-3-ol = 6:1:1. VOC stock solutions (0.1% v/v aqueous solution) were mixed proportionally and diluted 10³-fold to simulate early spoilage VOC concentrations. For induction experiments, 90 μL of bacterial culture was aliquoted into each well of a 96-well black microplate (Corning, USA), and 10 μL of mixed VOC solution was added, making the total system volume 100 μL. Control wells received 10 μL of sterile water.

Fluorescence Measurement

After mixing, samples were taken at time points 0 h (before treatment), 2 h, 4 h, 6 h, and 12 h to detect fluorescence intensity and OD600, calculating normalized fluorescence values. A multifunctional microplate reader (FlexStation 3, Molecular Devices, USA) was used to measure RFP fluorescence intensity (excitation wavelength 584 nm, emission wavelength 607 nm) and OD₆₀₀ to correct for cell density. Each group had three biological replicates, with two technical replicates each.

Data Processing and Statistical Analysis

Calculation of normalized fluorescence value:

![Experimental Results Diagram](https://static.igem.wiki/teams/5879/results/img15.webp)

Using the NormFluo of the CK group at corresponding time points as baseline, calculate induction fold change (FC):

![Experimental Results Diagram](https://static.igem.wiki/teams/5879/results/img16.webp)

Each time point included three independent biological replicates, with results expressed as mean ± standard deviation (SD). One-way ANOVA was used to compare differences in FC across time points, followed by Tukey's post-hoc test, with significance threshold set at p<0.05.

#### Results

The results showed that under mixed VOC treatment, the induction fold change of the PgrpE-mRFP reporter strain exhibited a continuous upward trend over time (Figure X). At 2 hours post-treatment, FC was approximately 1.3-fold, indicating the system could detect VOC signals within a short time. By 4 hours, FC increased to 2.6-fold; reaching 3.2-fold at 6 hours, and maintaining around 3.5-fold at high levels after 10 hours. This demonstrates that PgrpE has rapid response and high signal amplification effects to mixed VOC signals, further supporting its selection as the superior promoter.

Therefore, through testing with individual VOCs such as 1-octanol and their mixtures, the fluorescence signal mediated by grpE was significantly stronger than lasI, recA, and soxS, and could generate sustained responses to mixtures, confirming it as a superior VOC-responsive promoter.

![Experimental Results Diagram](https://static.igem.wiki/teams/5879/results/img17.webp)

Figure 13 Response fold changes of the PgrpE reporter strain to mixed VOCs at different time points. According to VOC composition distribution, the mixed VOCs had phenylethyl alcohol:1-octanol:1-octen-3-ol = 6:1:1.

#### Discussion

High Sensitivity of PgrpE to Mixed VOCs

The experimental results show that the induction fold change of the PgrpE-mRFP reporter system rapidly increased within a short time under VOC mixture treatment, peaking around 12 hours. This indicates that PgrpE can not only respond to individual VOCs but also maintain high comprehensive sensitivity in mixed gas environments.

Application Significance

The results demonstrate that the PgrpE reporter strain can generate detectable response signals within 1-2 hours and maintain detection effectiveness for a considerable duration, making it highly suitable for use as an early spoilage monitoring sensor. In practical fruit transportation or storage scenarios, the detection cycle could be set at 2-4 hours to achieve real-time early warning.

# II. Construction and Optimization of the Violacein Production System

## 2.1 Establishment of the Absorption Spectrum and Standard Curve for Violacein

### Objective

Before detecting violacein production levels, it is first necessary to determine its characteristic absorption wavelength and establish a standard curve for quantitative detection to ensure the accuracy and comparability of subsequent experimental data.

### Method

Chemically synthesized violacein standard was used to prepare a series of gradient concentration solutions (0–20 μg/mL) in 50% anhydrous ethanol. A spectrophotometer was used to scan the wavelength range of 300–700 nm to determine the maximum absorption peak wavelength of violacein in 50% anhydrous ethanol solution. The absorbance of each gradient concentration solution was measured at the maximum absorption wavelength, and a concentration-absorbance standard curve was plotted and linearly fitted.

![2.1 Establishment of the Absorption Spectrum and Standard Curve for Violacein Diagram](https://static.igem.wiki/teams/5879/results/img18.webp)

Figure 10 Establishment of the Absorption Spectrum and Standard Curve for Violacein

#### Result

The results indicate that violacein has a maximum absorption peak at 575 nm, and there is a good linear relationship between its absorbance and concentration. The linear regression equation is: Y = 1.733X + 0.08867 (R² = 0.99), where A represents the absorbance value and C represents the concentration (μg/mL).

![2.1 Establishment of the Absorption Spectrum and Standard Curve for Violacein Diagram](https://static.igem.wiki/teams/5879/results/img19.webp)

Figure 11 Scanning Spectrum and Standard Curve of Violacein in 50% Anhydrous Ethanol Solution

#### Discussion

The established standard curve can be used for quantitative determination of violacein concentration in subsequent experiments. The high correlation coefficient (R² = 0.99) of the linear fit indicates that this method has good accuracy and reproducibility, meeting the requirements for quantitative analysis.

## 2.2 Analysis of Violacein Production in Engineered Bacteria

### Objective

This experiment aims to evaluate the violacein synthesis capability and dynamic changes in engineered bacteria during fermentation when using violacein as a reporter gene. Due to its distinct advantages in visible color output and quantitative detection, violacein holds potential as a signal output in VOC (volatile organic compound) response systems. Therefore, by analyzing the relationship between violacein production and bacterial growth, the stability and visualization effect of this reporter gene expression in engineered bacteria can be assessed, providing experimental evidence for the design and optimization of subsequent VOC sensing systems.

### Methods

#### Construction of the Violacein-Producing Engineered Strain

The violacein biosynthesis gene cluster (BCG) comprising *vioAB* and *vioCDE* was synthesized. A constitutively expressed promoter PJ23100 and a medium-strength RBS B0034 were added upstream to drive gene expression. Codon optimization was performed for *E. coli*, and EcoRI, XbaI, SpeI, and PstI restriction sites were eliminated to comply with the RFC#10 standard. The cluster was then cloned into the pSB1A3 vector via EcoRI and XbaI, yielding the recombinant plasmid pSB1A3-vioABCDE. The recombinant plasmid was transformed into *E. coli* BL21 as previously described.

![2.2 Analysis of Violacein Production in Engineered Bacteria Diagram](https://static.igem.wiki/teams/5879/results/img20.webp)

1. Culture ConditionsThe engineered strain harboring the violacein synthesis genes was inoculated into M9 medium containing glucose as the carbon source in conical flasks and cultured at 37°C with 220 rpm shaking in a constant-temperature incubator. Samples were taken at various time points during fermentation (1, 3, 8, 12, 24, 36, 48, and 60 h) for determination of dry cell weight (DCW) and violacein production.
2. Violacein ExtractionTo ensure complete recovery of violacein, ethanol-based extraction was performed separately on the cells and culture supernatant:
3. Cell Fraction: After centrifugation to collect the cells, an appropriate amount of anhydrous ethanol was added. Ultrasonication (40 kHz, 10 min) was used to disrupt the cell walls and release violacein, followed by centrifugation to collect the supernatant.
4. Supernatant Fraction: An equal volume of anhydrous ethanol was directly added to the culture supernatant, mixed thoroughly, and centrifuged to collect the ethanol extract containing violacein.The two extracts were combined for unified quantitative analysis.

#### Quantitative Detection of Violacein

A spectrophotometer was used to measure the absorbance of violacein at 575 nm, and its concentration (mg/L) was calculated using the standard curve. All experiments were performed in triplicate, and the average values were calculated.

![2.2 Analysis of Violacein Production in Engineered Bacteria Diagram](https://static.igem.wiki/teams/5879/results/img21.webp)

### Results

(1) Biomass Dynamics

The dry cell weight (DCW) increased slowly within the first 8 hours (from 0.05 g/L to 0.12 g/L), indicating an adaptation phase and early exponential growth phase. After 12 hours, the culture entered a rapid growth phase, reaching 0.85 g/L at 24 hours and peaking at 1.1–1.2 g/L during 36–48 hours. The biomass stabilized after 60 hours.

(2) Violacein Production Changes

Violacein production was low in the initial phase (1–8 h), ranging from 0.02 to 0.05 mg/L. As the biomass increased rapidly, violacein production rose significantly between 12 and 24 hours, reaching 0.17 mg/L at 24 hours. It continued to increase to approximately 0.23–0.25 mg/L during 36–48 hours and remained at a plateau (around 0.24 mg/L) at 60 hours, closely aligning with the trend of biomass changes.

(3) Synchronization of Product and Biomass

The calculation of violacein/DCW revealed that violacein synthesis increased synchronously with biomass growth throughout the fermentation process, with the highest specific productivity (around 0.20 mg/g) observed during 24–36 hours. This synchronization indicates that violacein expression is directly coupled with the growth of the engineered bacteria, with no significant lag, demonstrating efficient synthesis of the reporter gene and good compatibility with host metabolism.

![2.2 Analysis of Violacein Production in Engineered Bacteria Diagram](https://static.igem.wiki/teams/5879/results/img22.webp)

Figure 16 Expression of *vioABCDE* driven by the J23100 promoter and B0034 RBS in BL21 using the pSB1A3 vector. DCW: dry cell weight. The medium used was M9 complete medium supplemented with glucose.

### Discussion

Violacein, as a reporter gene, demonstrates high efficiency, synchronization, and visualizability, making it suitable for real-time detection and result presentation in VOC response systems. This study provides a solid experimental foundation for subsequent system integration.

## 2.3 Glycerol Enhancement of Violacein Production

### Objective

As the final visual output signal in the VOC response system, the yield of violacein directly affects signal visibility and detection sensitivity. Carbon source composition plays a critical role in metabolic flux distribution and the synthesis of secondary metabolites. To further improve violacein production, this experiment compared the synthesis of violacein in engineered bacteria cultured in M9 minimal medium supplemented with either 5% glucose or 5% glycerol as the primary carbon source. By analyzing the impact of different carbon sources on violacein yield, the most suitable carbon source supplementation strategy was identified to provide a more efficient and intuitive signal output for subsequent VOC response systems.

### Methods

(1) Cultural Conditions

The engineered strain containing the violacein synthesis gene cluster (*vioABCDE*) was used as the experimental subject. Two culture systems were established:

① 5% glucose group;

② 5% glycerol group.

All cultures were incubated at 37°C with shaking at 220 rpm, with a culture volume of 50 mL.

(2) Violacein Extraction and Quantification

Samples were collected after 24 hours of cultivation. Violacein was extracted from both cells and culture supernatant using the previously established ethanol extraction method:

* Cell fraction: Cells were collected by centrifugation, and intracellular violacein was released by adding anhydrous ethanol followed by ultrasonic disruption.
* Supernatant fraction: Culture supernatant was directly mixed with an equal volume of anhydrous ethanol for extraction.The combined extracts were measured for absorbance at 575 nm using a spectrophotometer, and violacein concentration was calculated based on a standard curve.

(3) Data Processing

The methodology for data processing was consistent with the violacein quantification described in Section 2.3. All experiments were performed in triplicate, with mean values taken as final results and subjected to statistical significance analysis.

### Results

The experimental results demonstrated that different carbon sources significantly influenced violacein synthesis (Table 2, Figure 3). Under 5% glucose cultivation, violacein production ranged from 0.132 to 0.162 mg/L, with an average of 0.146 mg/L. In contrast, under 5% glycerol cultivation, violacein production significantly increased, ranging from 0.245 to 0.323 mg/L, with an average of 0.281 mg/L—approximately 1.9 times higher than under glucose conditions.

![2.3 Glycerol Enhancement of Violacein Production Diagram](https://static.igem.wiki/teams/5879/results/img23.webp)

Figure 17 Comparison of violacein content in BL21-Pj23100-RB0034-VioABCDE after 24 hours of cultivation with different carbon sources.

### Discussion

Supplementation with 5% glycerol significantly enhanced violacein production in the engineered bacteria, achieving nearly a 2-fold increase compared to the 5% glucose group, while also improving the stability and intuitiveness of the visual output. Therefore, glycerol is a more suitable carbon source choice for the subsequent construction of VOC-responsive sensing systems.

## 2.4 Analysis of VOC Response by PgrpE-VioABCDE Reporter Strain

### Objective

This experiment aimed to verify whether the *PgrpE* promoter can drive the expression of the *vioABCDE* violacein synthesis pathway upon detecting volatile organic compounds (VOCs) released during strawberry spoilage, thereby producing visible color changes. By integrating *PgrpE* with the *vioABCDE* pathway to construct a complete reporter system, we sought to transform invisible spoilage signals into a visual purple output. This approach aims to provide an instrument-free, low-cost, and real-time method for monitoring strawberry spoilage during cold chain transportation and storage.

### Methods

#### (1) Strain Construction and Cultivation

The composite part PgrpE-vioABCDE was constructed by combining the validated PgrpE promoter with a standard strong ribosome binding site (RBS, BBa\_B0034) and the violacein biosynthetic gene cluster (vioABCDE). This construct was cloned into the high-copy-number plasmid pSB1A3.

The recombinant plasmid was subsequently transformed into Escherichia coli BL21(DE3) cells. Transformed strains were cultured in Luria-Bertani (LB) liquid medium supplemented with 100 μg/mL ampicillin at 37°C with shaking at 250 rpm.

#### (2) VOC Preparation and Treatment

To simulate volatile organic compounds (VOCs) released during early strawberry spoilage, a mixture of three major spoilage-related compounds - phenylethyl alcohol, 1-octanol, and 1-octen-3-ol - was prepared in a 6:1:1 ratio, reflecting the actual volatile profile during spoilage processes.

In experimental setups, different volumes of the VOC mixture (10 μL, 20 μL, 50 μL, and 100 μL) were added to a 96-well plate, followed by inoculation with 90 μL of bacterial culture at the mid-logarithmic growth phase (OD₆₀₀ ≈ 0.6). Each concentration was tested with three biological replicates.

#### (3) Sampling and Analysis

Samples were collected at 0, 6, 12, 18, and 24-hour time points. Violacein was extracted using ethanol extraction followed by ultrasonic cell disruption.

Absorbance was measured at 570 nm, and violacein concentrations were determined using a pre-established standard calibration curve.

Control experiments were performed using bacterial cultures grown under identical conditions without VOC exposure.

![2.4 Analysis of VOC Response by PgrpE-VioABCDE Reporter Strain Diagram](https://static.igem.wiki/teams/5879/results/img24.webp)

Figure 18. Experimental workflow of the PgrpE-VioABCDE reporter strain

### Results

![2.4 Analysis of VOC Response by PgrpE-VioABCDE Reporter Strain Diagram](https://static.igem.wiki/teams/5879/results/img25.webp)

Figure 19. Response of PgrpE-VioABCDE reporter strain to VOCs.(A) Effect of different VOC concentrations on violacein production；(B) Visual observation of violacein accumulation under different VOC concentrations

Experimental results demonstrated that the PgrpE-vioABCDE reporter strain produced significant violacein accumulation upon VOC detection, with yields showing positive correlation with both VOC concentration and exposure duration (Figure19-A).

Post-incubation, cultures exposed to 100 μL VOCs exhibited deep purple coloration, the 50 μL group showed moderate purple, while the 10 μL and 20 μL groups displayed light purple coloration. Control groups remained colorless (Figure B). These findings validate the PgrpE reporter system as an effective visual detection platform for spoilage signals.

### Discussion

This study systematically validates the application feasibility of the PgrpE promoter coupled with the violacein biosynthetic pathway (vioABCDE) for early detection of strawberry spoilage:

* Sensitivity: The *PgrpE* promoter detected spoilage signals at VOC concentrations as low as 10 μL and effectively drove vioABCDE expression, demonstrating detection capability during initial spoilage stages.
* Dose and Time Dependency: Violacein production increased proportionally with higher VOC concentrations and extended exposure times, following characteristic dose-response kinetics. This indicates that PgrpE activation levels directly correlate with environmental spoilage signal intensity, enabling spoilage progression assessment.
* Visualization Advantage: The colorimetric output enables direct naked-eye detection without requiring sophisticated instrumentation, making it particularly suitable for resource-limited scenarios including cold chain logistics and storage facilities.
* Application Prospects: Considering current strawberry supply chain requirements, this reporter system shows potential for embedded packaging sensors and real-time transport container monitoring. The platform could be extended to monitor spoilage in other fruits such as blueberries and raspberries. Future developments could involve violacein pathway engineering or alternative output genes to achieve multiplexed color outputs or enhanced detection sensitivity.

In summary, thePgrpE-vioABCDE reporter strain successfully converts spoilage-associated VOC signals into visible colorimetric outputs, providing a cost-effective and user-friendly synthetic biology platform for fruit spoilage monitoring. This work establishes a foundation for developing advanced VOC-based food safety detection technologies.

# III. Construction and Validation of the Antifungal System

Throughout the post-harvest supply chain—including transportation and storage—strawberries are highly susceptible to fungal infections. Fungal metabolic activities can lead to rapid spoilage, resulting in significant economic losses. The VOC detection module developed in the earlier phase of this study enables real-time monitoring of spoilage-related volatile organic compounds (VOCs), providing early warning and risk indication. While this module successfully detects spoilage, feedback from Human Practices (HP) interviews revealed that detection alone does not fundamentally reduce spoilage rates or economic losses. Without intervention measures, early warning alone may still lead to fruit decay and waste.

To address this limitation, this study further designed an independent antifungal strategy as a supplement to the detection module:

After harvesting and before transportation or cold-chain storage, strawberry surfaces are treated by spraying two hydrolytic enzymes that degrade fungal cell walls—chitinase and β-1,3-glucanase.

The main structural components of fungal cell walls are chitin and β-1,3-glucan, which collectively maintain cell wall integrity and function.

* Chitinase cleaves chitin molecules, leading to the loosening and disintegration of the cell wall framework.
* β-1,3-Glucanase degrades β-1,3-glucan, disrupting the supportive network of the cell wall.

Through synergistic action, these two enzymes target and break down the core structure of the fungal cell wall at the molecular level, causing destabilization and functional failure. This ultimately inhibits fungal growth and may lead to cell death.

By applying these enzymes before transportation, a protective barrier can be established on the strawberry surface, effectively suppressing fungal infection. This approach reduces the spoilage rate of strawberries during transit and storage, thereby mitigating economic losses caused by fungal decay.

## 3.1 Expression Validation of Chitinase and Glucanase

### Experimental Objective

This section aims to construct expression systems for chitinase and β-1,3-glucanase, and validate the molecular weights of both enzymes against theoretical values through SDS-PAGE analysis. This ensures the correctness and reliability of the expressed products, while providing high-purity enzyme sources for subsequent antifungal activity assays and strawberry spray validation, thereby establishing a foundation for strawberry spoilage prevention experiments.

### Experimental Methods

#### 1. Construction of Antifungal Enzyme Expression Plasmids

To establish the antifungal enzyme expression system, this study selected two target genes capable of degrading major fungal cell wall components: chitinase and β-1,3-glucanase.

* The chitinase gene is approximately 1.2 kb in length, encoding a theoretical protein molecular weight of 44.6 kDa
* The β-1,3-glucanase gene spans approximately 1.0 kb, with a theoretical protein molecular weight of 42 kDa

To enhance their expression levels in *E. coli*, both genes underwent codon optimization for *E. coli* and were fused with a 6×His-tag at the 5′ terminus to facilitate subsequent protein purification via Ni-NTA affinity chromatography.

The optimized gene fragments were cloned into the pET28a(+) vector using NcoI and XhoI restriction enzyme sites, generating two recombinant plasmids:

* pET28a-Chitinase: for chitinase expression
* pET28a-Glucanase: for β-1,3-glucanase expression

The pET28a(+) vector contains a T7 promoter that enables high-level transcription in the host strain *E. coli* BL21(DE3).

* In BL21(DE3), the T7 RNA polymerase gene is controlled by the lacUV5 promoter and remains repressed under normal conditions
* The lacI repressor binds to the lacUV5 promoter, preventing T7 RNA polymerase transcription
* Upon addition of IPTG (isopropyl-β-D-thiogalactopyranoside), IPTG mimics lactose and binds to lacI, causing its dissociation from the lacUV5 promoter and lifting repression
* The derepressed lacUV5 promoter then drives T7 RNA polymerase expression, which subsequently binds to the T7 promoter on the plasmid, strongly activating downstream target gene transcription and enabling high-level protein expression

Following plasmid construction, double restriction enzyme digestion and Sanger sequencing were performed to verify correct gene insertion and sequence integrity.

![3.1 Expression Validation of Chitinase and Glucanase Diagram](https://static.igem.wiki/teams/5879/results/img26.webp)

Figure 17. Construction of pET28a-Chitinase vector

![3.1 Expression Validation of Chitinase and Glucanase Diagram](https://static.igem.wiki/teams/5879/results/img27.webp)

Figure 18. Construction of pET28a-Glucanase vector

#### 2. Transformation and Positive Clone Screening

Recombinant plasmids were introduced into *E. coli* BL21(DE3) competent cells via chemical transformation.

* The transformation mixture was spread on LB plates containing 50 μg/mL kanamycin and incubated at 37°C overnight
* Single colonies were picked the next day for colony PCR verification
* After confirming positive clones, glycerol stocks (25% glycerol) were prepared and stored long-term at -80°C

#### 3. Protein Expression and IPTG Induction

Pre-culture

* Verified positive clones were inoculated into 10 mL LB liquid medium containing kanamycin (50 μg/mL)
* Cultures were shaken at 37°C, 220 rpm overnight

Main Culture and Induction

* Overnight cultures were transferred to 50 mL fresh LB medium (with kanamycin) at 1% inoculation ratio
* Cultures were grown until OD₆₀₀ = 0.6–0.8 (logarithmic growth phase)
* IPTG was added to a final concentration of 1 mM for induction, while the temperature was reduced to 30°C to promote soluble protein expression
* Induction continued for 12 hours before cell harvest

#### 4. Protein Extraction and Purification

Cell Harvest and Resuspension

* After IPTG induction, cells were collected by centrifugation at 8000 g for 10 min
* Supernatant was discarded, and cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, containing 500 mM NaCl)

Ultrasonic Disruption

* Resuspended cells were placed on ice and disrupted by ultrasonication: 3 sec ON / 5 sec OFF, for 15 min total

Supernatant Collection

* Lysates were centrifuged at 12000 g for 15 min, and supernatants were collected as crude protein extracts

Ni-NTA Affinity Purification

* Supernatants were loaded onto Ni-NTA affinity columns, utilizing His-tag binding to nickel ions for target protein capture
* Target proteins were eluted using an imidazole concentration gradient:
* 20 mM: Remove non-specifically bound proteins
* 50 mM: Remove weakly bound contaminating proteins
* 200 mM: Elute target proteins
* Elution fractions were collected for subsequent SDS-PAGE analysis

#### 5.SDS-PAGE Electrophoresis Analysis Gel Formulation

* Separating Gel (12%):
* 30% acrylamide: 4.0 mL
* Tris-HCl (pH 8.8): 2.5 mL
* 10% SDS: 0.1 mL
* ddH₂O: 3.4 mL
* 10% APS: 100 μL
* TEMED: 10 μL
* Stacking Gel (5%):
* 30% acrylamide: 1.3 mL
* Tris-HCl (pH 6.8): 2.5 mL
* 10% SDS: 0.1 mL
* ddH₂O: 6.0 mL
* 10% APS: 50 μL
* TEMED: 5 μL

Sample Preparation and Loading

* 20 μL of protein solution was mixed with SDS loading buffer and denatured by heating in a 95°C water bath for 5 min.
* Prepared samples and molecular weight standard proteins were loaded simultaneously.

Electrophoresis and Staining

* Electrophoresis was performed at 120 V until the bromophenol blue front reached the bottom of the gel.
* After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 for 1 hour.
* Destaining was performed using a methanol/acetic acid destaining solution until the background was clear and target bands were visible.

Protein Size Verification

By comparing the positions of target bands with the molecular weight standard, the molecular weights of the expressed proteins were confirmed to match theoretical values:

* Chitinase: ~44.6 kDa
* β-1,3-Glucanase: ~42 kDa

![3.1 Expression Validation of Chitinase and Glucanase Diagram](https://static.igem.wiki/teams/5879/results/img28.webp)

Figure 19. Protein purification workflow

### Experimental Results

SDS-PAGE results are shown in Figure 20: After induction, a single clear band for RmChi44 chitinase appeared at 46.66 kDa, consistent with its theoretical molecular weight.

MoGluB β-1,3-glucanase showed the expected band at 45.57 kDa, with good expression levels.

These results indicate that both target proteins were efficiently and solubly expressed in BL21(DE3) and could be purified via His-tag binding to Ni-NTA.

![3.1 Expression Validation of Chitinase and Glucanase Diagram](https://static.igem.wiki/teams/5879/results/img29.webp)

Figure 20. SDS-PAGE of RmChi44 and MoGluB expressed in E. coli.（Lane M: Protein Marker；Lane 1: RmChi44 purified via Ni-NTA column；Lane 2: MoGluB purified via Ni-NTA column）

### Discussion

This experiment successfully established prokaryotic expression systems for chitinase and β-1,3-glucanase, with SDS-PAGE confirming correct expression. These enzymes target chitin and β-1,3-glucan in fungal cell walls, respectively, and can synergistically degrade fungal cell wall structures, theoretically exhibiting significant antifungal and bactericidal effects.

The band results demonstrate that both enzymes can be highly expressed in soluble form in *E. coli*, indicating that the vector design and expression conditions are appropriate. This provides a reliable enzyme source for subsequent fungal inhibition assays and strawberry spoilage model experiments.

## 3.2 Functional Validation of Chitinase and Glucanase Expression

### Objezctive

This experiment aims to validate the *in vitro* broad-spectrum antimicrobial activity of purified chitinase and β-1,3-glucanase. Using the model fungus *Saccharomyces cerevisiae* as the target, the inhibitory effects of individual enzymes and their combination on fungal growth are evaluated. Since chitin and β-1,3-glucan are core structural components of the cell walls in most fungi (including strawberry spoilage pathogens such as *Botrytis cinerea*, *Rhizopus* spp., and *Penicillium* spp.), this study provides critical functional evidence for the subsequent application of these enzymes on strawberry surfaces to broadly prevent postharvest spoilage caused by various fungi.

### Methods

#### Strains and Enzyme Preparations:

* Target strain: *Saccharomyces cerevisiae* BY4741. Yeast was selected as the target organism because the main components of its cell wall (chitin and β-1,3-glucan) are highly similar to those of the primary pathogenic fungi responsible for postharvest strawberry spoilage. Furthermore, it is safe to handle, does not produce aerial spores, and is suitable for antimicrobial validation in a laboratory environment.
* Enzyme preparations: The purified RmChi44 chitinase (~44.6 kDa) and MoGluB β-1,3-glucanase (~42 kDa) obtained via Ni-NTA affinity chromatography in section 3.1 were used. The enzyme solutions were exchanged into PBS buffer (pH 6.0), and their concentrations were determined using the BCA method and standardized to 1 mg/mL.

#### Yeast Suspension Preparation:

* A single yeast colony was inoculated into YPD liquid medium and cultured overnight at 30°C with shaking at 200 rpm.
* Culture from the logarithmic growth phase was taken and subjected to serial gradient dilution with sterile PBS (pH 6.0). The final concentration was adjusted to approximately 1×10⁶ CFU/mL for subsequent use.

#### Antimicrobial Assay Procedure:

* 100 µL of the aforementioned yeast suspension was thoroughly mixed with 100 µL of enzyme solution from different treatment groups in a 1.5 mL microcentrifuge tube, resulting in a final volume of 200 µL per reaction. The following five experimental groups were set up:
* Group 1: Wild-type Control: 200 µL sterile PBS buffer (pH 6.0), without yeast suspension, used to confirm the absence of contamination from the medium and aseptic techniques.
* Group 2: Negative Control: 100 µL yeast suspension + 100 µL of a mixed enzyme solution that had been heat-inactivated by treatment at 95°C for 15 minutes.
* Group 3: Chitinase Group: 100 µL yeast suspension + 100 µL chitinase solution (final concentration 0.5 mg/mL).
* Group 4: Glucanase Group: 100 µL yeast suspension + 100 µL glucanase solution (final concentration 0.5 mg/mL).
* Group 5: Combination Group: 100 µL yeast suspension + 50 µL chitinase solution + 50 µL glucanase solution (final concentration of each enzyme 0.25 mg/mL, total enzyme concentration 0.5 mg/mL).
* All mixed systems were gently incubated in a shaker at 30°C for 2 hours.
* After incubation, the reaction mixture from each group was subjected to 10-fold serial dilution (10⁻¹, 10⁻², 10⁻³) with PBS.
* 100 µL from each dilution was spread onto YPD solid agar plates. Three parallel replicates were performed for each dilution.
* The plates were inverted and incubated in a constant temperature incubator at 30°C for 36-48 hours.

#### Data Collection and Analysis

Colonies grown on agar plates were counted to determine the number of colony-forming units per milliliter of the original suspension (CFU/mL).The CFU/mL value of the negative control group (heat-inactivated enzyme treatment) was designated as representing 100% survival, and the relative survival rate (%) of each treatment group was calculated accordingly. All experimental data were presented as the mean ± standard deviation (Mean ± SD) of three independent biological replicates. Statistical significance among groups was assessed using one-way analysis of variance (ANOVA), with *p* < 0.05 considered statistically significant.

![3.2 Functional Validation of Chitinase and Glucanase Expression Diagram](https://static.igem.wiki/teams/5879/results/img30.webp)

Figure 21. Workflow for the expression and functional verification of *chitinase* and *glucanase*.

### Results

Quantitative results of the antifungal assays clearly demonstrated the inhibitory effects of *chitinase*, *glucanase*, and their combination against *Saccharomyces cerevisiae* (Figure 22, Table 1).

In the negative control group (Group 2, heat-inactivated enzymes), the yeast colony count was 7 × 10⁷ CFU/mL, which was set as the baseline survival rate (100%). This confirmed that the non-enzymatic components of the preparation had no effect on yeast growth.

Treatment with chitinase alone (Group 3) reduced the viable cell count to 1 × 10⁷ CFU/mL, corresponding to a relative survival rate of 14.3%, indicating that *chitinase* alone effectively degraded the fungal cell wall and inhibited cell viability.

The glucanase-treated group (Group 4) exhibited even stronger antifungal activity, with the colony number reduced to 3 × 10⁶ CFU/mL and a relative survival rate of 4.3%.

The combined enzyme treatment (Group 5) produced the most pronounced inhibitory effect, decreasing the viable count to 1.4 × 10⁶ CFU/mL and yielding a relative survival rate of only 2.0%.

This inhibition was significantly greater than that observed with either single-enzyme treatment (*p* < 0.001), revealing a strong synergistic antifungal effect between *chitinase* and *glucanase*.

![3.2 Functional Validation of Chitinase and Glucanase Expression Diagram](https://static.igem.wiki/teams/5879/results/img31.webp)

Figure 22. Synergistic inhibition of *S. cerevisiae* by the combined action of *chitinase* and *glucanase.*

![3.2 Functional Validation of Chitinase and Glucanase Expression Diagram](https://static.igem.wiki/teams/5879/results/img32.webp)

Table 1. Fungistatic activity of *chitinase* and *glucanase* against *S. cerevisiae.*

*Note: CFU = colony-forming unit. Relative survival rates were calculated based on the viable count of Group 2. Statistical significance was determined using one-way ANOVA; ### p < 0.001 vs. Group 2.*

### Discussion

Through rigorous quantitative analysis, this study successfully verified the effectiveness of the enzyme formulation developed in this project.

#### Efficacy validation:

The data clearly demonstrated that both purified enzymes possess strong in vitro antifungal activity, effectively degrading the cell wall of the model yeast and leading to a dramatic decrease in its survival rate. This provides direct evidence that functional proteins were successfully expressed and purified in *Escherichia coli.*

#### Synergistic effect:

The performance of the dual-enzyme combination was particularly remarkable. Its antifungal activity was not merely additive but exhibited a clear synergistic effect (*1 + 1 > 2*). This finding indicates that our strategy—simultaneously targeting two key structural components of fungal cell walls (*chitin* and *β-1,3-glucan*)—achieves a more thorough and efficient disruption of cell wall integrity. Such a design represents a highly effective and rational engineering approach.

#### Application potential:

Given the similarity in cell wall composition between *Saccharomyces cerevisiae* and pathogenic fungi that infect strawberries, these results strongly support the development of this enzyme mixture as a broad-spectrum and efficient biological preservative. Its application in postharvest strawberry treatment is expected to markedly suppress fungal infection and reduce spoilage losses.

Moreover, this response module seamlessly integrates with the previously developed VOC-sensing and *violacein*-based reporting modules, together forming a complete synthetic biology system that encompasses sensing, reporting, and responding. This integrated design perfectly embodies the engineering-driven spirit of iGEM—using synthetic biology to provide practical, real-world solutions.

### Conclusion

In the sensing module, screening of four VOC-responsive promoters (*PsoxS*, *PlasI*, *PrecA*, and *PgrpE*) revealed that the *PgrpE* promoter exhibited the highest induction fold and signal stability in response to multiple spoilage-associated VOCs (1-octanol, phenylethyl alcohol, and 1-octen-3-ol), making it the core sensing element for subsequent system construction.

In the reporting module, the combination of *PgrpE* with the *vioABCDE* chromogenic gene cluster successfully achieved a visual conversion of spoilage signals. Experimental results demonstrated that this system could generate a significant color change even at very low VOC concentrations, with color intensity positively correlated with both VOC concentration and exposure time—indicating a highly sensitive and intuitive detection capability.

In the response module, two antifungal proteins—*chitinase* and *β-1,3-glucanase*—were successfully constructed and expressed. SDS–PAGE analysis confirmed that their molecular weights matched theoretical values, and functional validation further demonstrated a significant synergistic antifungal effect of the enzyme combination, reducing fungal (*S. cerevisiae*) survival to below 2%.

Overall, the three modules form a functional synthetic biology loop that progresses from spoilage signal recognition (Sensing) to visual spoilage reporting (Reporting) and finally to fungal inhibition and prevention (Responding).

This integrated system provides a feasible technological framework for real-time detection and prevention of postharvest spoilage in agricultural products. Beyond strawberries, it also lays the foundation for future applications in food safety monitoring and the development of intelligent packaging systems.

{% endblock %}