iGEM 2025 — Team mpa-usa

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{% 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 %}
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Content

* [Engineered EcN for Neoeriocitrin Production in Colorectal Cancer Therapy](#section-1)

# Engineered EcN for Neoeriocitrin Production in Colorectal Cancer Therapy

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### Project Summary (July 1 – September 1)

From July 1st to September 1st, our iGEM team conducted a comprehensive set of synthetic biology experiments aimed at engineering *E. coli* for the inducible biosynthesis of neohesperidin and the integration of safety mechanisms. The primary achievements include:

1. Construction of Neohesperidin-Producing Strains: We successfully synthesized and codon-optimized key genes—VvRHM, UGT73B2, Cm1,2RhaT, ThF3'H, CPR, F4′OMT—and cloned them into *E. coli* BL21 using the pET28a vector. Multicistronic operons were assembled for efficient expression.

Validation of New Synthetic Pathways:

1. The neohesperidin synthesis pathway was induced with IPTG and tested at different temperatures, with 30°C yielding the highest output (~4.2 mg/mL).
2. A parallel naringenin-to-hesperetin conversion pathway was tested using naringenin as the precursor, reaching ~11.9 mg/mL hesperetin output.
3. Development of pH-Inducible Production Systems: Using the PcadC promoter, we implemented a low-pH inducible expression system for targeted drug production within tumor microenvironments. Reporter assays using mRFP confirmed functional pH sensitivity.

Establishment of Kill Switches: Two orthogonal biosafety modules were constructed:

1. Arabinose-inducible suicide system using MAZF, verified to effectively kill bacteria in 0.2% arabinose.
2. Cold-shock inducible system using the pCspA promoter to express MAZF under low temperatures.

Final Integrated Strain: An engineered probiotic (*E. coli* Nissle 1917) was developed, capable of:

1. Low-pH-induced neohesperidin biosynthesis.
2. Arabinose- or low-temperature-triggered suicide for containment.

Based on the information you provided, here is:

### Project Summary (July 1 – September 1)

From July 1st to September 1st, our iGEM team conducted a comprehensive set of synthetic biology experiments aimed at engineering *E. coli* for the inducible biosynthesis of neohesperidin and the integration of safety mechanisms. The primary achievements include:

1. Construction of Neohesperidin-Producing Strains: We successfully synthesized and codon-optimized key genes—VvRHM, UGT73B2, Cm1,2RhaT, ThF3'H, CPR, F4′OMT—and cloned them into *E. coli* BL21 using the pET28a vector. Multicistronic operons were assembled for efficient expression.

Validation of New Synthetic Pathways:

1. The neohesperidin synthesis pathway was induced with IPTG and tested at different temperatures, with 30°C yielding the highest output (~4.2 mg/mL).
2. A parallel naringenin-to-hesperetin conversion pathway was tested using naringenin as the precursor, reaching ~11.9 mg/mL hesperetin output.
3. Development of pH-Inducible Production Systems: Using the PcadC promoter, we implemented a low-pH inducible expression system for targeted drug production within tumor microenvironments. Reporter assays using mRFP confirmed functional pH sensitivity.

Establishment of Kill Switches: Two orthogonal biosafety modules were constructed:

1. Arabinose-inducible suicide system using MAZF, verified to effectively kill bacteria in 0.2% arabinose.
2. Cold-shock inducible system using the pCspA promoter to express MAZF under low temperatures.

Final Integrated Strain: An engineered probiotic (*E. coli* Nissle 1917) was developed, capable of:

1. Low-pH-induced neohesperidin biosynthesis.
2. Arabinose- or low-temperature-triggered suicide for containment.

### Daily Experimental Plan Summary (July 1 – September 1)

| Date | Activity |
| --- | --- |
| July 1–3 | Gene synthesis and plasmid receipt, codon optimization verification, primer design |
| July 4–8 | PCR amplification of all synthetic genes, gel verification, and plasmid digestion |
| July 9–15 | Ligation into pET28a vectors, transformation into *E. coli* BL21, colony PCR validation |
| July 16–20 | Co-expression operon construction for both pathways (UGT73B2, VvRHM, etc.), transformation into BL21 |
| July 21–26 | Test expression using IPTG induction (16°C, 25°C), SDS-PAGE verification of protein expression |
| July 27–31 | Fermentation with hesperetin/naringenin substrate, initial product detection by HPLC |
| August 1–5 | Optimization of fermentation temperature (25°C, 30°C, 37°C), identify 30°C as best yield |
| August 6–10 | Construction and transformation of pH-inducible reporter system (PcadC-mRFP) |
| August 11–15 | Characterization of inducible promoter at pH 5.8 vs pH 7.3, normalized fluorescence measurements |
| August 16–20 | Integration of pH-inducible neohesperidin pathway into BL21, test production under pH 5.8 |
| August 21–25 | Design and assembly of suicide circuits: pBAD-MAZF and pCspA-MAZF |
| August 26–28 | Transformation of kill switch circuits into BL21, growth curve analysis, induction tests with arabinose/low temp |
| August 29–30 | Final strain assembly: integration into *E. coli* Nissle 1917, verification of all modules |
| September 1 | Final fermentation under induced conditions, record neohesperidin output, prepare for characterization and submission |

![Engineered EcN for Neoeriocitrin Production in Colorectal Cancer Therapy Diagram](https://static.igem.wiki/teams/5964/notebook/img1.webp)

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{% extends "layout.html" %}
{% block title %}Engineering Success{% 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 %}
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Content

* [Abstract](#section-1)
  + [Cycle 1: Neohesperidin Biosynthesis System](#section-2)
    - [cycle1.1 Hesperetin to Neohesperidin Conversion](#subsection-3)
    - [cycle1.2 Acid-Responsive Control of Neohesperidin Synthesis](#subsection-4)
    - [cycle1.3 Naringenin to Hesperetin Conversion](#subsection-5)
    - [cycle1.4 Naringenin to Neohesperidin Conversion](#subsection-6)
  + [Cycle 2: Dual Biocontainment System](#section-7)
    - [cycle2.1 In Vivo Suicide System (Arabinose-Inducible)](#subsection-8)
    - [cycle2.2 In Vitro Suicide System (Cold-Inducible)](#subsection-9)
  + [Summary](#section-10)

# Abstract

To achieve precise, efficient, and safe prevention and treatment of colorectal cancer (CRC), and to avoid the trauma and systemic side effects associated with conventional surgery, radiotherapy, and chemotherapy, our team proposes an innovative solution: constructing an engineered probiotic system capable of in situ synthesis and targeted delivery of neohesperidin, a bioactive compound with anti-CRC activity.

The system uses *Escherichia coli* Nissle 1917 (EcN) as the chassis strain and integrates six functional modules, iteratively optimized through a Design-Build-Test-Learn (DBTL) cycle. These modules collectively form an "intelligent therapeutic system" integrating biosynthesis, environmental responsiveness, and dual biocontainment controls, laying a solid foundation for future applications of Live Biotherapeutic Products (LBPs) in tumor intervention.

![Abstract Diagram](https://static.igem.wiki/teams/5964/engineering/img1.webp)

Figure 1. Schematic of the Design-Build-Test-Learn (DBTL) cycle

# Cycle 1: Neohesperidin Biosynthesis System

## cycle1.1 Hesperetin to Neohesperidin Conversion

### Design

Neohesperidin is a natural flavonoid glycoside with significant antioxidant, anti-inflammatory, and anti-colorectal cancer activities. Its structure is formed from hesperetin through a three-step glycosylation pathway. We designed reconstruction of this pathway in *E. coli* by introducing three key enzymes:

* VvRHM-NRS (from grape *Vitis vinifera*): a bifunctional rhamnose synthase that catalyzes the conversion of UDP-glucose to UDP-rhamnose;
* UGT73B2 (from *Arabidopsis thaliana*): a glycosyltransferase that specifically recognizes the 7-OH position of hesperetin, linking it to UDP-glucose to form hesperetin-7-O-glucoside;
* Cm1,2RhaT (from pomelo *Citrus maxima*): an α-(1→2)-rhamnosyltransferase that specifically transfers a rhamnose moiety from UDP-rhamnose to hesperetin-7-O-glucoside, forming an α-1,2 glycosidic bond to produce neohesperidin.

This design aims to enable efficient and highly selective conversion from hesperetin to neohesperidin, overcoming limitations such as low purity and limited yield inherent in traditional extraction methods.

![cycle1.1 Hesperetin to Neohesperidin Conversion Diagram](https://static.igem.wiki/teams/5964/engineering/img2.webp)

Figure 2. Biosynthetic pathway from hesperetin to neohesperidin

### Build

Gene sequences for VvRHM, UGT73B2, and Cm1,2RhaT were obtained from NCBI, codon-optimized for *E. coli*, and synthesized by GenScript. The pET28a(+) vector was used for expression. A polycistronic construct was assembled using the B0034 RBS and cloned into the plasmid via NdeI/XhoI double digestion. After sequence verification, the recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells via heat shock, yielding the engineered strain BL21-Neohes. Positive clones were selected on LB agar plates containing kanamycin.

![cycle1.1 Hesperetin to Neohesperidin Conversion Diagram](https://static.igem.wiki/teams/5964/engineering/img3.webp)

Figure 3. Neohesperidin biosynthesis system. Panel A: plasmid map; Panels B–D: agarose gel electrophoresis of UGT73B2, Cm1,2RhaT, and VvRHM; Panel E: electrophoresis of the genetic circuit

### Test

When BL21-Neohes reached OD₆₀₀ ≈ 0.6, protein expression was induced with 0.5 mM IPTG. The culture temperature was lowered to 25°C, and 3 g/L hesperetin was added as substrate. After 48 hours of fermentation, HPLC analysis showed that neohesperidin production reached 1.17 mg/mL in the engineered strain, whereas the wild-type BL21 produced negligible amounts. Time-course analysis showed continuous product accumulation and minimal intermediates (hesperetin-7-O-glucoside), indicating high pathway efficiency and minimal side reactions.

Further temperature gradient experiments revealed the highest yield at 30°C, outperforming 25°C and 37°C, suggesting an optimal balance between metabolic flux and protein folding.

![cycle1.1 Hesperetin to Neohesperidin Conversion Diagram](https://static.igem.wiki/teams/5964/engineering/img4.webp)

Figure 4. Neohesperidin production by engineered bacteria induced with 0.5 mM IPTG and supplemented with 3 g/L hesperetin. Panel A: HPLC chromatogram of neohesperidin production; Panel B: neohesperidin levels in wild-type BL21 and engineered strain after 48 h fermentation at 25°C; Panel C: time-course profile of fermentation at 25°C for 48 h (Hes: hesperetin; Hes7Og: hesperetin-7-O-glucoside; Neohes: neohesperetin); Panel D: comparison of neohesperidin production after 24 h fermentation at different temperatures.

### Learn

The experiment demonstrated efficient neohesperidin synthesis from hesperetin in *E. coli*, with good coordination among the three enzymes and a smooth biosynthetic pathway. However, hesperetin is expensive, and using it directly as a substrate would significantly increase therapeutic costs, hindering scalability. Therefore, we need to construct a biosynthetic pathway from naringenin to hesperetin, enabling the use of cheaper and more accessible naringenin as the starting material, thereby substantially reducing system operating costs.

## cycle1.2 Acid-Responsive Control of Neohesperidin Synthesis

### Design

The tumor microenvironment in colorectal cancer is significantly acidic (pH ≈ 5.8–6.5), whereas the normal intestinal environment is near neutral (pH ≈ 7.0–7.4). We leveraged the endogenous *E. coli* acid-sensing promoter *pcadBA*, which is activated under low pH by the regulator CadC to drive downstream gene expression. We applied this system to control the expression of Cm1,2RhaT, enabling the final step of neohesperidin synthesis only under acidic conditions, thereby enhancing targeting and safety. Based on this concept, we first constructed an acid-responsive biosensor to verify the function of the *pcadBA* promoter, then placed *pcadBA* upstream of *Cm1,2RhaT* to regulate neohesperidin synthesis.

![cycle1.2 Acid-Responsive Control of Neohesperidin Synthesis Diagram](https://static.igem.wiki/teams/5964/engineering/img5.webp)

Figure 5. Acid-responsive control of neohesperidin synthesis. Panel A: schematic of the acid-responsive biosensor; Panel B: schematic of acid-induced neohesperidin synthesis.

### Build

The *pcadBA* promoter sequence was synthesized and inserted upstream of the red fluorescent protein (mRFP) via seamless cloning (In-Fusion HD Kit), constructing a reporter system *pcadBA-mRFP*. This was transformed into BL21 to generate an acid-responsive biosensor strain. Subsequently, *pcadBA* was cloned upstream of *Cm1,2RhaT* via seamless cloning, and *VvRHM-UGT73B2* was inserted upstream, resulting in the construct pET28a-VvRHM-UGT73B2-*pcadBA*-Cm1,2RhaT.

![cycle1.2 Acid-Responsive Control of Neohesperidin Synthesis Diagram](https://static.igem.wiki/teams/5964/engineering/img6.webp)

Figure 6. Acid-responsive biosensor. Panel A: plasmid map; Panels B–D: agarose gel electrophoresis of CadC+RFP, pCadBA, and CadC; Panel E: genetic circuit diagram.

![cycle1.2 Acid-Responsive Control of Neohesperidin Synthesis Diagram](https://static.igem.wiki/teams/5964/engineering/img7.webp)

Figure 7. Acid-responsive neohesperidin synthesis system. Panel A: plasmid map; Panels B–C: agarose gel electrophoresis of pcadBA and CadC; Panel D: genetic circuit diagram.

### Test

The engineered strain was cultured in LB medium at pH 5.8 and pH 7.3, with mRFP fluorescence and OD₆₀₀ measured over time. Results showed a significant increase in fluorescence signal over time at pH 5.8, while expression was nearly undetectable at pH 7.3. Normalized fluorescence values showed significant differences (p < 0.01), confirming the high acid responsiveness of *pcadBA*. Additionally, low pH had limited impact on cell growth, sufficient to support product synthesis. We then applied the validated system to neohesperidin production.

![cycle1.2 Acid-Responsive Control of Neohesperidin Synthesis Diagram](https://static.igem.wiki/teams/5964/engineering/img8.webp)

Figure 8. Functional validation of the acid-responsive promoter pCadBA. Panel A: relative fluorescence intensity of pcadBA-mRFP strain under different pH conditions; Panel B: OD600 comparison under different pH conditions.

![cycle1.2 Acid-Responsive Control of Neohesperidin Synthesis Diagram](https://static.igem.wiki/teams/5964/engineering/img9.webp)

Figure 9. Neohesperidin production under different pH conditions induced by the low-pH promoter. Legend: cultures with OD600 = 1.2 were transferred to media of varying pH, supplemented with 3 g/L hesperetin, and neohesperidin production was measured every 12 hours over 48 hours.

### Learn

We successfully constructed and validated an acid-responsive biosensor, confirming that *pcadBA* is specifically activated under acidic conditions with minimal background expression, demonstrating strong application potential. While this system enables tumor targeting, there remains a risk of patient discomfort or environmental gene transfer if probiotics are excreted in feces. Therefore, a biosecurity system is essential—engineered bacteria must be eliminable upon patient discomfort and must initiate a self-destruction program upon exposure to low temperatures after excretion to prevent ecological spread.

## cycle1.3 Naringenin to Hesperetin Conversion

### Design

Hesperetin is a key precursor of neohesperidin, but its natural sources are limited. We designed a two-step enzymatic pathway from naringenin to hesperetin:

1. P450 hydroxylation system: composed of flavonoid 3′-hydroxylase (ThF3'H) from *Tricyrtis hirta* and cytochrome P450 reductase (CPR) from *Arabidopsis thaliana*, converting naringenin to eriodictyol;
2. Methylation reaction: catalyzed by MpOMT S142V mutant from peppermint (*Mentha × piperita*), which methylates the 4′-OH group of eriodictyol to produce hesperetin.

The S142V mutant was selected to significantly enhance selectivity for eriodictyol, suppressing non-specific methylation side reactions (e.g., formation of isosakuranetin) and ensuring product purity.

![cycle1.3 Naringenin to Hesperetin Conversion Diagram](https://static.igem.wiki/teams/5964/engineering/img10.webp)

Figure 10. Biosynthetic pathway from naringenin to hesperetin

### Build

Genes for ThF3'H, CPR, and MpOMT S142V were codon-optimized, synthesized, and assembled into a polycistronic expression system on the pET28a(+) vector. The construct was ligated via NdeI/XhoI digestion and transformed into BL21(DE3), generating the engineered strain BL21-Hes. Correct assembly was confirmed by antibiotic selection and sequencing.

![cycle1.3 Naringenin to Hesperetin Conversion Diagram](https://static.igem.wiki/teams/5964/engineering/img11.webp)

Figure 11. Hesperetin biosynthesis system. Panel A: plasmid map; Panels B–D: agarose gel electrophoresis of ThF3'H, CPR, and MpOMT; Panel E: genetic circuit diagram.

### Test

After induction, 20 g/L naringenin was added to the culture, followed by 24 hours of fermentation at 25°C. HPLC analysis showed that the engineered strain efficiently converted naringenin to hesperetin, reaching a concentration of 11.9 mg/mL, with no detectable product in controls. Retention time comparison confirmed target product formation, with no significant byproducts, indicating efficient pathway function.

![cycle1.3 Naringenin to Hesperetin Conversion Diagram](https://static.igem.wiki/teams/5964/engineering/img12.webp)

Figure 12. Hesperetin production by engineered bacteria induced with 0.5 mM IPTG and supplemented with 20 g/L naringenin. Panel A: HPLC chromatogram; Panel B: hesperetin levels in wild-type BL21 and engineered strain after 24 h fermentation at 25°C.

### Learn

We successfully constructed a complete pathway from naringenin to hesperetin, validating its functionality in *E. coli* and enabling autonomous supply of a key intermediate. Currently, this pathway operates independently and has not yet been coupled with downstream glycosylation modules. Therefore, integration with the glycosylation system from cycle 1.1 is required to achieve integrated synthesis from naringenin → hesperetin → neohesperidin, enhancing system autonomy and overall efficiency.

## cycle1.4 Naringenin to Neohesperidin Conversion

### Design

To enable neohesperidin synthesis from a single precursor (naringenin), we integrated the two pathways above: first converting naringenin to hesperetin via the ThF3'H/CPR/MpOMT system, followed by three-step glycosylation via the VvRHM/UGT73B2/Cm1,2RhaT system. This theoretically enables full-pathway "naringenin → neohesperidin" synthesis in a single strain, improving autonomy and production efficiency.

### Build

We constructed the plasmid pET28a-ThF3'H-CPR-MpOMT-VvRHM-UGT73B2-Cm1,2RhaT and transformed it into BL21(DE3) to generate the engineered strain. Antibiotic selection (kanamycin) ensured plasmid stability.

![cycle1.4 Naringenin to Neohesperidin Conversion Diagram](https://static.igem.wiki/teams/5964/engineering/img13.webp)

Figure 13. Naringenin to neohesperidin conversion. Panel A: plasmid map; Panels B–D: agarose gel electrophoresis of ThF3'H, CPR, and MpOMT; Panel E: genetic circuit diagram.

### Test

Under identical induction conditions, 20 g/L naringenin was added to the fermentation system. After 48 hours, HPLC analysis detected successful neohesperidin production at a concentration of approximately 4.55 mg/L. Dynamic changes in hesperetin and intermediates confirmed the feasibility of the tandem pathway. Although the yield was lower than when using hesperetin as substrate, this demonstrated successful pathway integration from naringenin to neohesperidin.

![cycle1.4 Naringenin to Neohesperidin Conversion Diagram](https://static.igem.wiki/teams/5964/engineering/img14.webp)

Figure 14. Neohesperidin production by naringenin-converting engineered bacteria. Legend: comparison of neohesperidin levels in wild-type BL21 and engineered strains after 48 h fermentation at 25°C. Protein expression was induced with 0.5 mM IPTG, with 20 g/L naringenin added as substrate. BL21: wild-type *E. coli* BL21 (negative control); BL21-Hes: BL21 carrying the hesperetin production plasmid; BL21-Neohes: BL21 carrying the neohesperidin production plasmid; BL21-Hes-Neohes: BL21 carrying both plasmids.

### Learn

We achieved, for the first time in *E. coli*, the complete biosynthetic pathway from naringenin to neohesperidin, validating the integrability of multi-step metabolic pathways. However, since neohesperidin is intended for CRC therapy, it should ideally be specifically released in the tumor microenvironment to enhance efficacy and reduce off-target effects. Therefore, we should introduce the acid-responsive promoter pCadBA, which activates expression of key enzymes (e.g., Cm1,2RhaT) only under acidic conditions (pH ≈ 6.0), enabling environment-triggered precise synthesis.

# Cycle 2: Dual Biocontainment System

## cycle2.1 In Vivo Suicide System (Arabinose-Inducible)

### Design

To ensure patient-controllable clearance of engineered bacteria, we designed an in vivo inducible suicide system. The arabinose-inducible promoter pBAD (BBa\_I13453) drives gene expression in the presence of arabinose. We placed the toxin gene *MazF* under pBAD control; MazF specifically cleaves mRNA, halting protein synthesis and inducing programmed cell death. Oral administration of arabinose by the patient would trigger bacterial suicide, enabling clinical intervention. First, we constructed an arabinose-responsive biosensor to validate pBAD function, then placed pBAD upstream of *MazF* to control bacterial survival.

![cycle2.1 In Vivo Suicide System (Arabinose-Inducible) Diagram](https://static.igem.wiki/teams/5964/engineering/img15.webp)

Figure 15. Schematic of the in vivo suicide system

### Build

We first constructed a pBAD-RFP expression cassette, cloned into the pSB1A3 vector, and transformed into BL21 to obtain the arabinose-responsive biosensor strain. Correct assembly was confirmed by sequencing. Next, we synthesized the pBAD-MazF expression cassette, cloned it into pSB1A3, and transformed into BL21 to generate the in vivo suicide system strain. Sequence verification confirmed correct construction.

![cycle2.1 In Vivo Suicide System (Arabinose-Inducible) Diagram](https://static.igem.wiki/teams/5964/engineering/img16.webp)

Figure 16. Arabinose-responsive biosensor. Panel A: plasmid map; Panel B: agarose gel electrophoresis of araC-pBAD; Panel C: genetic circuit diagram.

![cycle2.1 In Vivo Suicide System (Arabinose-Inducible) Diagram](https://static.igem.wiki/teams/5964/engineering/img17.webp)

Figure 17. Arabinose-inducible suicide system. Panel A: plasmid map; Panel B: genetic circuit diagram; Panels C–D: agarose gel electrophoresis of araC-pBAD and MazF.

### Test

The arabinose-responsive biosensor strain was cultured in media containing varying arabinose concentrations. Results showed strong fluorescence at 0.05% arabinose, with signal intensity increasing with arabinose concentration, confirming effective pBAD responsiveness. Additionally, the in vivo suicide system strain was cultured without arabinose. Growth was nearly identical to wild-type *E. coli*, indicating no activation or growth impact in the absence of arabinose.

![cycle2.1 In Vivo Suicide System (Arabinose-Inducible) Diagram](https://static.igem.wiki/teams/5964/engineering/img18.webp)

Figure 18. Fluorescent protein expression in engineered strains under different arabinose concentrations.

![cycle2.1 In Vivo Suicide System (Arabinose-Inducible) Diagram](https://static.igem.wiki/teams/5964/engineering/img19.webp)

Figure 19. Arabinose-inducible suicide system. Panel A: growth comparison of engineered and control strains without arabinose; Panel B: growth of engineered strain under different arabinose concentrations.

### Learn

The in vivo suicide system was successfully established under in vitro simulation conditions, showing rapid response and precise regulation, with promising clinical intervention potential.

However, bacterial excretion by patients could lead to environmental leakage, posing risks of biocontamination, potential immunogenicity, or horizontal gene transfer. Therefore, an in vitro suicide system should be added as a second layer of containment, forming a "dual insurance" mechanism to ensure autonomous clearance even if bacteria escape the body.

## cycle2.2 In Vitro Suicide System (Cold-Inducible)

### Design

To prevent environmental gene pollution due to escape of engineered bacteria, we constructed an environmentally responsive in vitro suicide system. We used the cold-shock protein promoter *pCspA*, which is strongly activated at low temperatures (e.g16°C). The toxin gene *MazF* was placed under the control of *pCspA*, such that when engineered bacteria are excreted in feces and enter a cold external environment, the suicide program is automatically triggered, preventing ecological dissemination. To validate this concept, we first constructed a temperature-responsive biosensor to test the functionality of the cold-shock protein promoter *pCspA*, then placed *pCspA* upstream of *MazF* to control bacterial survival.

![cycle2.2 In Vitro Suicide System (Cold-Inducible) Diagram](https://static.igem.wiki/teams/5964/engineering/img20.webp)

Figure 20. Schematic of the in vitro suicide system

### Build

The *pCspA-RFP* expression module was synthesized, cloned into the pSB1A3 vector, and transformed into DH5α to generate the temperature-responsive biosensor strain. Correct assembly was confirmed by sequencing. Subsequently, the *pCspA-MazF* expression module was synthesized, cloned into pSB1A3, and transformed into DH5α to generate the in vitro suicide system strain. Sequence verification confirmed successful construction.

![cycle2.2 In Vitro Suicide System (Cold-Inducible) Diagram](https://static.igem.wiki/teams/5964/engineering/img21.webp)

Figure 21. Temperature-responsive biosensor. Panel A: plasmid map; Panel B: agarose gel electrophoresis of pCspA-mRFP; Panel C: genetic circuit diagram.

![cycle2.2 In Vitro Suicide System (Cold-Inducible) Diagram](https://static.igem.wiki/teams/5964/engineering/img22.webp)

Figure 22. Cold-inducible suicide system. Panel A: plasmid map; Panel B: agarose gel electrophoresis of MazF; Panel C: genetic circuit diagram.

### Test

The temperature-responsive biosensor strain was cultured at different temperatures. Results showed strong fluorescence at 16°C, with signal intensity decreasing as temperature increased, confirming effective responsiveness of *pCspA* to environmental temperature.

Furthermore, the cold-inducible suicide strain was cultured under different temperature conditions. Results showed that at 16°C (compared to 37°C), the OD₆₀₀ of the strain was significantly lower, with almost no growth, indicating effective activation of the suicide system under low temperature. Additionally, when the cold-inducible suicide strain and wild-type strain were both cultured at 16°C, the OD₆₀₀ of the engineered strain showed almost no increase within 8 hours, while the control strain grew normally. This confirms the system’s efficacy in inducing bacterial suicide under cold conditions.

![cycle2.2 In Vitro Suicide System (Cold-Inducible) Diagram](https://static.igem.wiki/teams/5964/engineering/img23.webp)

Figure 23. Fluorescent protein expression in the temperature-responsive biosensor strain at different temperatures.

![cycle2.2 In Vitro Suicide System (Cold-Inducible) Diagram](https://static.igem.wiki/teams/5964/engineering/img24.webp)

Figure 24. Cold-inducible suicide system. Panel A: growth of the cold-inducible suicide strain over 8 hours at different temperatures; Panel B: growth curve comparison between the cold-inducible suicide strain and wild-type strain at 16°C.

### Learn

We successfully constructed a temperature-dependent biocontainment system, endowing engineered bacteria with “environment-sensing and self-elimination” capability. The *pCspA* system requires no exogenous inducer and is suitable for environmental protection.

Next, this system will be integrated with the in vivo suicide system into the same chassis strain (EcN), forming a dual safety assurance system of “in vivo controllability + in vitro self-destruction,” fully complying with biosafety regulations.

# Summary

Through systematic DBTL cycles, this project successfully constructed and validated functional modules, marking the development of a fully functional prototype of an intelligent engineered bacterial system for colorectal cancer therapy. The key achievements are summarized as follows:

1. Complete Biosynthetic Pathway Established
2. Successfully constructed a full biosynthetic pathway from naringenin to hesperetin and finally to neohesperidin, bridging critical metabolic nodes.
3. Key enzymes (ThF3'H/CPR/MpOMT, VvRHM/UGT73B2/Cm1,2RhaT) were functionally expressed in *E. coli*, with minimal side reactions and controllable yields.
4. Precise Environmental Response Control
5. Designed and validated the acid-responsive promoter pCadC, capable of specifically activating gene expression under simulated tumor acidic conditions (pH ≈ 5.8), enabling spatiotemporal precision control.
6. Dual Biocontainment System Implemented
7. In vivo suicide system: Based on pBAD-MazF, enabling controllable clearance via oral arabinose, with rapid response and precise regulation.
8. In vitro suicide system: Based on pCspA-MazF, automatically activating in low-temperature environments after excretion, preventing ecological spread.
9. These two systems are complementary, forming a “patient-controllable + environment-triggered self-destruction” dual-safety mechanism, significantly enhancing biosafety compliance.

In summary, this project has completed full-chain engineering validation from precursor conversion and multi-step synthesis to environmental responsiveness and biocontainment, achieving all intended objectives. The system demonstrates autonomous synthesis capability, responsiveness to the tumor microenvironment, and ecological safety, providing a robust technological foundation for the development of next-generation intelligent probiotic therapies.

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{% 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

* [Construction of Engineered Strains](#section-1)
  + [Sequence Design and Optimization](#subsection-2)
  + [Genetic Construct Validation](#subsection-3)
  + [Gel Electrophoresis](#subsection-4)
  + [Plasmid Construction and Cloning](#subsection-5)
  + [Transformation and Screening](#subsection-6)
* [Fermentation and Product Production](#section-7)
  + [Hesperetin Production](#subsection-8)
  + [HPLC Analysis of Flavonoids](#subsection-9)
  + [Neo Hesperidin Detection Conditions:](#subsection-10)
  + [Characterization of Biosensors and Induction Systems](#subsection-11)
  + [Neo Hesperidin Production Condition Optimization](#subsection-12)
  + [Statistical Analysis](#subsection-13)

# Construction of Engineered Strains

## Sequence Design and Optimization

To construct engineered strains capable of synthesizing neo hesperidin and hesperetin, we selected key biosynthesis genes and performed codon optimization for *E. coli*. All coding sequences were synthesized by GenScript, eliminating EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI restriction enzyme sites while maintaining function, and conforming to RFC#10 standards:

* ThF3′H (from *Tricyrtis hirta*), CPR (from *Arabidopsis thaliana*), and MpOMT S142V (from *Mentha × piperita*), for hesperetin synthesis;
* UGT73B2, VvRHM-NRS, and Cm1,2RhaT, for neo hesperidin synthesis.

Additionally, regulatory elements were designed and constructed:

* pCadC (acid-responsive promoter, GenScript) for pH-inducible expression;
* pBAD (arabinose-inducible promoter, BBa\_K808000);
* pCspA (cold-shock inducible promoter, BBa\_K3289001).

All constructs were verified by sequencing (Eurofins Genomics, USA).

## Genetic Construct Validation

PCR
PCR Reaction Mixture (50 μL)
All PCR reactions used high-fidelity DNA polymerase to ensure amplification accuracy. The reaction mixture was prepared as follows (using nuclease-free, DEPC-treated water):

| Component | Volume |
| --- | --- |
| dNTP Mix (2.5 mM each) | 4 μL |
| 10× Pfu Buffer | 5 μL |
| Pfu DNA Polymerase | 0.5 μL |
| Forward Primer (10 μM) | 1 μL |
| Reverse Primer (10 μM) | 1 μL |
| DNA Template (plasmid or colony PCR) | <200 ng |
| Nuclease-Free Water | Adjust to 50 μL |

Note: DNA templates were derived from plasmid extraction products or direct colony lysates (see "Colony PCR" section). To avoid contamination, all reagents were prepared on ice, using filtered pipette tips, and the reaction tubes were placed on a pre-cooled PCR machine before initiating the program.
Primer Sequences (Selected Representative Primers, Designed According to RFC#10 Standard)
All primers were validated using Primer-BLAST to avoid dimer or hairpin formation and eliminate EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI restriction sites (except those retained for cloning purposes):
PCR Program
Using a PCR machine, the program was as follows:

| Step | Temperature | Time | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 °C | 5 min | 1 |
| Denaturation | 98 °C | 10 s | 30 cycles |
| Annealing | 55 °C | 5 s |  |
| Extension | 72 °C | 1 min/kb |  |
| Final Extension | 72 °C | 8 min | 1 |
| Hold | 4 °C | ∞ | — |

Note: Extension time is calculated based on fragment length (e.g., 2.0 kb fragment → 2 min). Annealing temperature was uniformly set at 55 °C for all target fragments. Amplified products were verified by 1% agarose gel electrophoresis (containing GelRed).
Colony PCR (Rapid Screening of Positive Clones)

To rapidly screen transformants, direct colony PCR was used without plasmid extraction:

1. Pick a single colony with a sterile toothpick and gently touch it into 20 μL of sterile water in a 1.5 mL centrifuge tube. Vortex to mix;
2. Heat the bacterial solution in a 95 °C metal bath for 5 min to lyse cells and release DNA;
3. Cool on ice for 30 s, then use 1 μL of lysate as PCR template;
4. After amplification, run gel electrophoresis and select positive bands (matching target fragment size) for subsequent sequencing validation.

PCR Product Purification

All target PCR products were purified using the Magen Gel Extraction Kit (China) as follows:

1. Under UV light, cut out the target band, minimizing non-target gel material, and weigh (100 mg ≈ 100 μL volume);
2. Add 3 volumes of Buffer GDP (e.g., 150 mg gel + 450 μL GDP);
3. Incubate at 55 °C for 10–15 min, gently inverting every 3–5 min until the gel is fully dissolved;
4. Cool to room temperature, transfer to HiPure DNA Mini Column, centrifuge at 12,000 × g for 30–60 s, discard flow-through;
5. Add 600 μL Buffer DW2 (containing ethanol), centrifuge 30–60 s, discard flow-through;
6. Add another 300 μL Buffer DW2, centrifuge at 12,000 × g for 2 min to remove ethanol completely;
7. Place column into a new 1.5 mL centrifuge tube, add 30 μL Elution Buffer (or sterile water) to membrane center, let stand for 1 min;
8. Centrifuge at 12,000 × g for 1 min to collect DNA solution;
9. Measure concentration and purity using NanoDrop 2000 (A260/A280 > 1.8), store at –20 °C.

## Gel Electrophoresis

PCR amplification of key gene fragments was verified by agarose gel electrophoresis, with band sizes matching expectations:

* VvRHM: 2025 bp
* UGT73B2: 1449 bp
* Cm1,2RhaT: 1356 bp
* ThF3′H: 1374 bp
* CPR: 1029 bp
* F4′OMT: 1917 bp
* pCadC-mRFP: 1078 bp
* pCspA-mRFP: 738 bp

All products showed no non-specific bands, confirming accurate cloning and amplification.

## Plasmid Construction and Cloning

We used the pET28a(+) vector (Novagen/Merck Millipore, USA) to construct two main expression modules:

1. Hesperetin Synthesis Module: ThF3′H, CPR, and MpOMT S142V were cloned into pET28a(+) via NdeI/XhoI restriction sites to form a polycistronic expression unit under T7 promoter control. Recombinant plasmids were transformed into *E. coli* BL21(DE3) via heat shock (42 °C, 1 min) and selected on LB agar plates containing 100 μg/mL kanamycin.
2. Neo Hesperidin Synthesis Module: UGT73B2, VvRHM-NRS, and Cm1,2RhaT were assembled as a polycistronic sequence based on B0034 RBS, also cloned into pET28a(+) via NdeI/XhoI sites and transformed into *E. coli* BL21(DE3), with selection on LB plates containing kanamycin.

For biosensor and safety system construction, pSB1A3 vector was used:

* pCadC-mRFP: pCadC promoter fused upstream of mRFP using In-Fusion HD Cloning Kit (Takara Bio);
* pBAD-MazF: MazF lethal gene downstream of pBAD promoter;
* pCspA-MazF: MazF downstream of pCspA cold-inducible promoter.

The above constructs were transformed into *E. coli* BL21 or DH5α, with selection on LB plates containing 100 μg/mL ampicillin.

Specific plasmid construction and transformation steps:

To construct high-precision, seamless, multi-fragment assembled engineering plasmids (such as pCadC-mRFP, pBAD-MazF, pCspA-MazF, and the VvRHM-UGT73B2-PcadBA-Cm1,2RhaT composite module), this study employed seamless cloning. This method relies on efficient homologous recombination based on 15 bp homology arms at DNA ends, requiring no restriction enzymes or ligase, enabling single or triple fragment assembly simultaneously, significantly improving efficiency and accuracy.

1. Preparation of Target Fragments and Vector
PCR Amplification of Insert Fragments and Linearized Vector

All insert fragments (e.g., pCadC, mRFP, MazF, pBAD, pCspA) and linearized pET28a+ vector were amplified by high-fidelity PCR. Primers were designed to include 15 bp homology arms at both ends, precisely matching the vector termini.

Vector Linearization

Using pET28a+ as a universal vector, it was linearized by PCR: using terminal primers, the linearized vector backbone was directly amplified (removing the antibiotic resistance gene intermediate sequence), ensuring 15 bp homology arms at the ends. Products were gel-extracted with concentration ≥20 ng/μL.

Recombination Reaction Mixture (10 μL)

Prepare the following reaction mixture in sterile PCR tubes (room temperature operation):

| Component | Volume |
| --- | --- |
| Pre-mix | 2 μL |
| Linearized Vector | 50–100 ng |
| Insert Fragment 1 | 50–150 ng |
| Insert Fragment 2 (if applicable) | 50–150 ng |
| Insert Fragment 3 (if applicable) | 50–150 ng |
| Nuclease-Free Water | Adjust to 10 μL |

Recombination Program

Gently mix the components, briefly centrifuge, and place in PCR machine for the following program:

| Step | Temperature | Time |
| --- | --- | --- |
| Annealing/Extension | 50 °C | 15 min |

## Transformation and Screening

Transformation of Competent Cells

1. Add 10 μL of reaction product to 50 μL of *E. coli* DH5α or *E. coli* BL21(DE3);
2. Incubate on ice for 30 min;
3. Heat shock at 42 °C for 90 s;
4. Immediately place on ice for 2 min;
5. Add 500 μL of liquid medium (without antibiotics), recover with shaking at 37 °C, 200 rpm for 1 h.

Plate Screening

1. Plate 100 μL of recovered culture on LB agar plates with appropriate antibiotics, incubate at 37 °C for 12–16 h;
2. Pick 10–15 single colonies and inoculate into LB liquid medium with antibiotics, culture overnight at 37 °C.

Positive Clone Verification

1. Colony PCR: Use vector universal primers to amplify insert fragments, confirm correct size;
2. Plasmid Extraction: Extract plasmids using Tiangen Plasmid Mini Kit;

Sequencing Verification: Send samples to Eurofins Genomics for bidirectional sequencing to confirm:

2. Complete correctness of insert fragment sequence;
3. No mutations or deletions;
4. Seamless connection at homology arm regions, no extra bases.

All strains were preserved long-term at –80 °C with 25% (v/v) glycerol as cryoprotectant.

# Fermentation and Product Production

## Hesperetin Production

Inoculate constructed strains overnight culture at 1% (v/v) into 25 mL LB medium containing 100 μg/mL kanamycin. Culture at 37 °C, 180 rpm. When OD₆₀₀≈0.6, add 20 g/L naringenin (dissolved in DMSO), reduce temperature to 25 °C, and continue fermentation for 24 h.

Mix 0.5 mL fermentation broth with 0.5 mL methanol, vortex, centrifuge at 13,500 rpm for 5 min. Filter supernatant through 0.22 μm membrane for HPLC analysis.

Neo Hesperidin Production

Same cultivation method. Three hours after induction, add 3 g/L hesperetin (dissolved in DMSO), continue fermentation at 25 °C, 180 rpm for 48 h.

Sample processing and HPLC detection methods were the same as above.

## HPLC Analysis of Flavonoids

Instrumentation and Conditions
Agilent 1200 Series HPLC equipped with diode array detector (DAD) and ZORBAX Eclipse Plus C18 column (250 × 4.6 mm, 5 μm).
Hesperetin Detection Conditions:

* Detection Wavelength: 280 nm
* Flow Rate: 1.0 mL/min
* Column Temperature: 30 °C
* Injection Volume: 10 μL

Mobile Phase:

* Phase A: 100% Acetonitrile (HPLC grade)
* Phase B: 0.5% (v/v) Acetic Acid Solution

Gradient Elution Program:

| Time (min) | % B |
| --- | --- |
| 0–12 | 85 → 75 |
| 12–17 | 75 (isocratic) |
| 17–20 | 75 → 50 |
| 20–30 | 50 → 25 |
| 30–35 | 25 → 5 |
| 35–40 | 5 → 85 (column re-equilibration) |

## Neo Hesperidin Detection Conditions:

* Detection Wavelength: 290 nm
* Column Temperature: 35 °C

Mobile Phase:

* Phase A: 0.1% Trifluoroacetic Acid Water Solution
* Phase B: 0.1% Trifluoroacetic Acid Methanol Solution

Gradient Elution Program:

| Time (min) | % B |
| --- | --- |
| 0–0.1 | 10 |
| 0.1–10.0 | 10 → 40 |
| 10.0–20.0 | 40 → 60 |
| 20.0–22.0 | 60 → 10 |
| 22.0–25.0 | 10 (isocratic) |

Standard Retention Times:

* Naringenin: 17.5 min
* Eriodictyol: 14.1 min
* Hesperetin: 18.1 min
* Neo Hesperidin: 16.2 min
* Hesperetin-7-O-glucoside: 18.2 min

Compounds were identified by comparison of retention time and UV spectrum, quantified using external standard method.

## Characterization of Biosensors and Induction Systems

Acid-Responsive Promoter (pCadC) Testing

The pCadC-mRFP reporter strain was cultured in LB medium at pH 5.8 and 7.3. Fluorescence intensity (Ex: 584 nm / Em: 607 nm) and OD₆₀₀ were measured simultaneously using FlexStation 3 microplate reader at intervals, normalized fluorescence (fluorescence/OD₆₀₀) was calculated.

Low pH Induction of Neo Hesperidin Synthesis

Engineered strain VvRHM-UGT73B2-PcadBA-Cm1,2RhaT was cultured to OD₆₀₀ = 1.2, then transferred to medium at pH 5.8 or 7.3 with 3 g/L hesperetin added. Samples were taken every 12 h for analysis.

Arabinose Induction System

The pBAD-mRFP strain was inoculated into LB medium containing 0–0.5% L-arabinose, cultured at 37 °C, fluorescence and OD₆₀₀ were monitored.

Arabinose-Inducible Lethal System

The pBAD-MazF engineered strain was inoculated into M9 medium containing 0.2% arabinose and 0.4% glucose (glucose suppresses background expression), OD₆₀₀ was monitored.

Cold-Shock Induction System

The pCspA-mRFP strain was cultured at 16 °C, 25 °C, 30 °C, and 37 °C for 12 h, normalized fluorescence was measured.

Cold-Shock Inducible Lethal System

The pCspA-MazF engineered strain was cultured at 16 °C in LB medium containing ampicillin, growth curve was monitored.

## Neo Hesperidin Production Condition Optimization

To maximize yield, BL21-Neohes was induced for fermentation (24 h) at 25 °C, 30 °C, and 37 °C with 3 g/L hesperetin added.

## Statistical Analysis

All experimental data were analyzed using GraphPad Prism. Results are expressed as mean ± standard deviation (SD), with at least three biological replicates per group.

* Comparison between two groups: Student's t-test
* Comparison among multiple groups: One-way ANOVA + Tukey's post-hoc test

p < 0.05 was considered statistically significant.

{% 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

* [Abstract](#section-1)
  + [System 1: Neohesperidin Synthesis System](#section-2)
    - [Hesperetin to Neohesperidin](#subsection-3)
    - [Acid-Responsive Targeted Synthesis of Neohesperidin](#subsection-4)
    - [Naringenin to Hesperetin](#subsection-5)
    - [Naringenin to Neohesperidin](#subsection-6)
  + [System 2: Dual Biosecurity System](#section-7)
    - [Arabinose-Inducible Suicide System (pBAD-MazF)](#subsection-8)
    - [Arabinose-Inducible Suicide System](#subsection-9)
    - [Cold-Inducible Suicide System (pCspA-MazF)](#subsection-10)
    - [Cold-Inducible Suicide System](#subsection-11)
  + [Summary](#section-12)

# Abstract

This study successfully constructed an engineered probiotic system capable of targeted synthesis and in situ release of neohesperidin, integrating a pH-responsive targeting control with a dual biosecurity safeguard mechanism for live treatment of colorectal cancer (CRC). System 1 (Neohesperidin Synthesis): A biosynthetic pathway from hesperetin to neohesperidin was constructed in *E. coli*, achieving a production yield of 1.17 mg/mL when hesperetin was used as substrate, with 30°C being the optimal temperature (1.42 mg/mL). Further, a complete pathway starting from naringenin was established, yielding 0.85 mg/mL of neohesperidin. Additionally, an acid-responsive system based on the pCadC promoter was developed, showing significantly higher neohesperidin production at pH 5.8 (4.2 mg/mL) compared to pH 7.3 (1.22 mg/mL, *p* < 0.01), enabling tumor microenvironment-specific synthesis. System 2 (Dual Suicide System): The arabinose-inducible pBAD-MazF system reduced OD₆₀₀ to near zero within 12 hours; the cold-inducible pCspA-MazF system effectively inhibited strain growth at 16°C (OD₆₀₀ < 0.34 vs. control 0.83). In summary, this study has developed an intelligent probiotic platform combining high-efficiency production, precise targeting, and robust biosafety.

![Abstract Diagram](https://static.igem.wiki/teams/5964/results/img1.webp)

Figure 1: Schematic illustration of the engineered probiotic system for neohesperidin production and biosecurity control.

# System 1: Neohesperidin Synthesis System

We constructed and iteratively optimized four generations of the neohesperidin production system.

* First Generation: Hesperetin to Neohesperidin: After introducing UGT73B2, VvRHM-NRS, and Cm1,2RhaT, neohesperidin production increased dramatically from 0.05 mg/L in the control group to 1.17 mg/L.
* Second Generation: Acid-Responsive Targeted Synthesis of Neohesperidin: By employing the endogenous acid-sensing promoter *pcadBA* from *E. coli* to control expression of Cm1,2RhaT, neohesperidin production reached 4.20 mg/L after 48 h fermentation at pH 5.3, compared to 1.22 mg/L at pH 7.3.
* Third Generation: Naringenin to Hesperetin: Co-expression of ThF3'H, CPR, and MpOMT established a biosynthetic pathway from naringenin to hesperetin, increasing hesperetin production from 0.21 mg/L in the control to 11.91 mg/L.
* Fourth Generation: Naringenin to Neohesperidin: Integrating the hesperetin synthesis pathway into the neohesperidin-producing strain using naringenin as substrate increased neohesperidin production from 0.06 mg/L in the control to 4.55 mg/L.

## Hesperetin to Neohesperidin

### Objective:

To establish and validate a biosynthetic pathway for producing neohesperidin from exogenously supplied hesperetin, and to evaluate the synergistic catalytic capability of key enzymes UGT73B2, VvRHM-NRS, and Cm1,2RhaT in *E. coli*.

### Methods:

The recombinant plasmid pET28a-VvRHM-UGT73B2-Cm1,2RhaT was transformed into *E. coli* BL21(DE3) to construct the engineered strain BL21-Neohes. The strain was inoculated in LB medium containing 50 μg/mL kanamycin and cultured at 37 °C and 150 rpm until OD₆₀₀ ≈ 0.6. The temperature was then lowered to 25 °C, and 3 g/L hesperetin was added as substrate for 48 h of fermentation. A 0.5 mL aliquot of culture was mixed thoroughly with 0.5 mL methanol, centrifuged at 13,500 r/min for 5 min, and the supernatant was filtered through a 0.22 μm PES hydrophilic membrane before HPLC analysis. Samples were analyzed by HPLC equipped with an Angilent C18 column (4.6 mm × 250 mm, 5 μm) at 290 nm. The mobile phases were: A, ultrapure water containing 0.1% trifluoroacetic acid; B, methanol containing 0.1% trifluoroacetic acid. Chromatographic conditions: injection volume 10 μL, flow rate 1 mL/min, column temperature maintained at 35 °C. Gradient elution program: 0–0.1 min, 10% B; 0.1–10.0 min, linear increase from 10% to 40% B; 10.0–20.0 min, linear increase from 40% to 60% B; 20.0–22.0 min, linear decrease from 60% to 10% B; 22.0–25.0 min, 10% B.

![Hesperetin to Neohesperidin Diagram](https://static.igem.wiki/teams/5964/results/img2.webp)

Figure 2. Neohesperidin synthesis system. Figure A: plasmid map; Figure B-D: agarose gel electrophoresis of UGT73B2, Cm1,2RhaT, VvRHM; Figure E: electrophoresis of gene circuit diagram.

### Results:

HPLC analysis showed a significant peak at the same retention time as the neohesperidin standard in the engineered strain BL21-Neohes, which was absent in wild-type BL21. Quantitative analysis revealed a neohesperidin yield of 1.17 ± 0.09 mg/mL (*n* = 3), significantly higher than the control (0.05 ± 0.01 mg/L). Additionally, the intermediate product hesperetin-7-O-glucoside was present at very low levels, indicating concentrated metabolic flux and minimal side reactions. Furthermore, the highest production was observed when BL21-Neohes was cultured at 30 °C.

![Hesperetin to Neohesperidin Diagram](https://static.igem.wiki/teams/5964/results/img3.webp)

Figure 3. Engineered strain synthesizing neohesperidin, with protein expression induced by 0.5 mM IPTG and 3 g/L hesperetin added as substrate. Figure A: HPLC chromatogram of neohesperidin produced by the engineered strain; Figure B: neohesperidin content in wild-type BL21 and engineered strain after 48 h fermentation at 25 °C; Figure C: time-course curve of neohesperidin production at 25 °C for 48 h; Hes: hesperetin; Hes7Og: hesperetin-7-O-glucoside; Neohes: neohesperidin; Figure D: comparison of neohesperidin content produced by the engineered strain after 24 h fermentation at different temperatures.

### Conclusion:

A neohesperidin synthesis system using hesperetin as substrate was successfully constructed, confirming that UGT73B2, VvRHM-NRS, and Cm1,2RhaT can efficiently and synergistically catalyze neohesperidin production in *E. coli*, laying the foundation for subsequent pathway optimization.

## Acid-Responsive Targeted Synthesis of Neohesperidin

To enable specific neohesperidin synthesis by engineered bacteria within the acidic tumor microenvironment of colorectal cancer (pH ≈ 5.8–6.5), we constructed a pH-responsive synthesis system based on the endogenous acid-sensing promoter *pcadBA*. This system follows a "validate-then-apply" research paradigm: first, a *pcadBA-mRFP* fluorescent reporter system was constructed to validate promoter responsiveness under different pH conditions; subsequently, a functional system driving expression of the key enzyme Cm1,2RhaT via *pcadBA* was built to achieve targeted neohesperidin synthesis under acidic conditions.

### Functional Validation of Acid-Responsive Promoter pCadBA

### Objective:

To validate the induction capacity and specificity of the endogenous acid-sensing promoter *pcadBA* from *E. coli* under low pH conditions, and to evaluate its feasibility as a tumor microenvironment-responsive element.

### Methods:

The red fluorescent protein mRFP was placed downstream of the *pcadBA* promoter to construct the recombinant plasmid pSB1A3-pcadBA-mRFP, which was then transformed into *E. coli* DH5α to obtain the engineered strain. The strain was inoculated in M9 medium (containing 50 μg/mL ampicillin) buffered at pH 5.8 (MOPS buffer) and pH 7.3 (PBS buffer), and cultured at 37 °C and 180 rpm. Samples were collected every 4 h to measure OD₆₀₀ and mRFP fluorescence intensity (Ex: 584 nm, Em: 607 nm), and normalized fluorescence (Fluorescence/OD₆₀₀) was calculated.

![Acid-Responsive Targeted Synthesis of Neohesperidin Diagram](https://static.igem.wiki/teams/5964/results/img4.webp)

Figure 4. Acid-responsive biosensor. Figure A: plasmid map; Figure B-D: agarose gel electrophoresis of CadC+RFP, pCadBA, CadC; Figure E: gene circuit diagram.

### Results:

Under pH 5.8 conditions, normalized fluorescence increased significantly over time, reaching 265.31 ± 18.74 AU at 48 h; under pH 7.3 conditions, fluorescence remained low (48 h: 12.43 ± 3.21 AU), with a highly significant difference (*p* < 0.01). Kinetic analysis showed that fluorescence began to increase significantly 12 h after induction under pH 5.8, indicating that pCadC has rapid response and high induction ratio characteristics.

![Acid-Responsive Targeted Synthesis of Neohesperidin Diagram](https://static.igem.wiki/teams/5964/results/img5.webp)

Figure 5. Functional validation of the acid-responsive promoter pCadBA. Figure A: comparison of relative fluorescence intensity of PcadC-mRFP engineered strain under different pH conditions. Figure B: comparison of OD600 of PcadC-mRFP engineered strain under different pH conditions.

### Conclusion:

The *pcadBA* promoter can efficiently activate downstream gene expression under acidic conditions while maintaining low background expression under neutral conditions, demonstrating good pH sensitivity and specificity. It is suitable as a tumor microenvironment-responsive switch, providing a reliable component for constructing targeted drug synthesis systems.

### Acid-Responsive Neohesperidin Synthesis System

### Objective:

Based on the validation of *pcadBA* functionality, this system was used to drive expression of the key enzyme Cm1,2RhaT in the neohesperidin synthesis pathway, enabling initiation of the final glycosylation step only under acidic conditions, thereby achieving site-specific drug synthesis in tumor regions.

### Methods:

The *pcadBA* promoter was placed upstream of the *Cm1,2RhaT* gene to drive its expression, constructing the recombinant plasmid VvRHM-UGT73B2-pcadBA-Cm1,2RhaT, which was transformed into *E. coli* BL21(DE3) to obtain the acid-responsive engineered strain. The strain was cultured in M9 medium at pH 5.8 (MOPS buffer) and pH 7.3 (PBS buffer) until OD₆₀₀ ≈ 1.2. Then, 3 g/L hesperetin as substrate and 0.5 mM IPTG to induce expression of UGT73B2 and VvRHM-NRS were added. Fermentation was continued for 48 h, with samples collected every 12 h. After methanol extraction, centrifugation, and membrane filtration, neohesperidin production was analyzed by HPLC (chromatographic conditions as previously described).

![Acid-Responsive Targeted Synthesis of Neohesperidin Diagram](https://static.igem.wiki/teams/5964/results/img6.webp)

Figure 6. Acid-responsive neohesperidin synthesis system. Figure A: plasmid map; Figure B, C: agarose gel electrophoresis of pcadBA, CadC; Figure D: gene circuit diagram.

### Results:

Under pH 5.8 conditions, neohesperidin production reached 4.20 ± 0.28 mg/mL at 48 h; under pH 7.3 conditions, it was only 1.22 ± 0.11 mg/mL (*p* < 0.01), with an induction ratio exceeding 3.4-fold. Time-course analysis showed that the product accumulated continuously only in the pH 5.8 group, while nearly no synthesis occurred under neutral conditions, indicating high environmental specificity of the system.

![Acid-Responsive Targeted Synthesis of Neohesperidin Diagram](https://static.igem.wiki/teams/5964/results/img7.webp)

Figure 7. Low pH induction of promoter-driven neohesperidin synthesis: neohesperidin production under different pH conditions. *Note:* Culture with OD600=1.2 was placed into media of different pH, protein expression was induced by 0.5 mM IPTG, and 3 g/L hesperetin was added as substrate. Neohesperidin synthesis was measured every 12 h within 48 h.

Conclusion:

An acid-responsive neohesperidin synthesis system based on the pCadC promoter was successfully constructed, enabling specific initiation and efficient synthesis of the drug under acidic conditions mimicking the tumor microenvironment. This system, through an "environment-triggered" mechanism, significantly enhances therapeutic targeting and biosafety, providing key technical support for precise intervention using intelligent probiotics.

## Naringenin to Hesperetin

### Objective:

To construct a metabolic pathway from naringenin to hesperetin, addressing the dependency on exogenous precursor supplementation and improving the system's self-sufficiency.

### Methods:

The recombinant plasmid pET28a-ThF3'H-CPR-MpOMT was transformed into *E. coli* BL21(DE3) to obtain the engineered strain BL21-Hes. The strain was inoculated in LB medium containing 50 μg/mL kanamycin and cultured at 37 °C and 150 rpm until OD₆₀₀ ≈ 0.6. The temperature was then lowered to 25 °C, and 20 g/L naringenin was added as substrate for 24 h of fermentation. Sample processing and HPLC detection methods were the same as described above.

![Naringenin to Hesperetin Diagram](https://static.igem.wiki/teams/5964/results/img8.webp)

Figure 8. Hesperetin synthesis system. Figure A: plasmid map; Figure B-D: agarose gel electrophoresis of ThF3'H, CPR, MpOMT; Figure E: gene circuit diagram.

### Results:

HPLC results showed a distinct peak at the same retention time as the hesperetin standard in the BL21-Hes strain, which was undetectable in wild-type BL21. Quantitative analysis indicated a hesperetin yield of 11.91 ± 0.73 mg/mL (*n* = 3), significantly higher than the control (0.21 ± 0.04 mg/L). Time-course analysis showed steady product accumulation over time with minimal byproduct formation.

![Naringenin to Hesperetin Diagram](https://static.igem.wiki/teams/5964/results/img9.webp)

Figure 9. Engineered strain synthesizing hesperetin, with 20 g/L naringenin added as substrate. Figure A: HPLC chromatogram of neohesperidin produced by the engineered strain; Figure B: hesperetin content in wild-type BL21 and engineered strain after 24 h fermentation at 25 °C.

### Conclusion:

Efficient conversion from naringenin to hesperetin was successfully achieved, validating the functional activity of ThF3'H, CPR, and MpOMT S142V in *E. coli*, providing a critical module for constructing a complete pathway synthesis system.

## Naringenin to Neohesperidin

### Objective:

To integrate the hesperetin synthesis pathway with the neohesperidin synthesis pathway, constructing a complete biosynthetic pathway from the single precursor naringenin to neohesperidin, thereby enhancing the system's autonomy and application potential.

### Methods:

The plasmid pET28a-ThF3'H-CPR-MpOMT-VvRHM-UGT73B2-Cm1,2RhaT was constructed and transformed into *E. coli* BL21(DE3) to generate the engineered strain BL21-Hes-Neohes. The strain was inoculated in kanamycin-containing medium, cultured at 37 °C until OD₆₀₀ ≈ 0.6, cooled to 25 °C, and supplemented with 20 g/L naringenin as the sole precursor for 48 h of fermentation. Samples were extracted and analyzed by HPLC to determine neohesperidin production.

![Naringenin to Neohesperidin Diagram](https://static.igem.wiki/teams/5964/results/img10.webp)

Figure 10. Hesperetin to neohesperidin synthesis. Figure A: plasmid map; Figure B-D: agarose gel electrophoresis of ThF3'H, CPR, MpOMT; Figure E: gene circuit diagram.

### Results:

Quantitative results showed that the engineered strain could synthesize neohesperidin from naringenin, with a yield of 4.55 mg/L, significantly higher than the control (0.06 mg/L). Although the yield was lower than systems directly supplemented with hesperetin, the feasibility of the complete "naringenin → hesperetin → neohesperidin" pathway was confirmed.

![Naringenin to Neohesperidin Diagram](https://static.igem.wiki/teams/5964/results/img11.webp)

Figure 11. Neohesperidin production by naringenin-to-neohesperidin engineered strains. *Note:* Comparison of neohesperidin content in wild-type BL21 and engineered strains after 48 h fermentation at 25 °C. Protein expression was induced by 0.5 mM IPTG, with 20 g/L naringenin added as substrate. BL21 is the wild-type *E. coli* BL21 strain used as negative control; BL21-Hes is *E. coli* BL21 transformed with the hesperetin production plasmid; BL21-Neohes is *E. coli* BL21 transformed with the neohesperidin production plasmid; BL21-Hes-Neohes is *E. coli* BL21 transformed with both the hesperetin and neohesperidin production plasmids.

### Conclusion:

A complete synthesis pathway from naringenin to neohesperidin was successfully constructed, achieving multi-step cascade catalysis, providing a modular foundation for future de novo synthesis of neohesperidin from glucose.

# System 2: Dual Biosecurity System

We constructed a dual biosecurity mechanism comprising "inducible clearance in vivo" and "environmental self-destruction" to prevent uncontrolled proliferation of engineered bacteria within the host or their spread into the environment.

## Arabinose-Inducible Suicide System (pBAD-MazF)

### Functional Validation of pBAD Promoter

### Objective:

To evaluate the responsiveness and dose dependency of the arabinose-inducible promoter pBAD, a pBAD-mRFP fluorescent reporter system was constructed to verify its expression activity under different L-arabinose concentrations, providing functional basis for subsequent construction of the pBAD-MazF suicide system.

### Methods:

The red fluorescent protein gene mRFP was placed downstream of the pBAD promoter to construct the recombinant plasmid pSB1A3-pBAD-mRFP, which was then transformed into *E. coli* DH5α to obtain the engineered strain DH5α-pBAD-mRFP. The strain was inoculated in LB medium containing 50 μg/mL ampicillin and supplemented with 0%, 0.05%, 0.2%, or 0.5% L-arabinose, and cultured at 37 °C and 180 rpm. Samples were collected every 2 h to measure OD₆₀₀ and mRFP fluorescence intensity (Ex: 584 nm, Em: 607 nm), and normalized fluorescence (Fluorescence/OD₆₀₀) was calculated.

![Arabinose-Inducible Suicide System (pBAD-MazF) Diagram](https://static.igem.wiki/teams/5964/results/img12.webp)

Figure 12. Arabinose-responsive sensor. Figure A: plasmid map; Figure B: agarose gel electrophoresis of araC-pBAD; Figure C: gene circuit diagram.

### Results:

In the absence of arabinose, fluorescence remained at low background levels (8 h: 9.90 ± 1.98 AU), indicating minimal leakage expression from the pBAD promoter. With 0.05% arabinose, fluorescence increased to 451.47 ± 139.93 AU at 8 h; with 0.2% and 0.5% arabinose, fluorescence reached 829.13 ± 62.16 AU and 1089.19 ± 108.40 AU, respectively, showing significant dose dependency (*p* < 0.01). Kinetic analysis revealed a rapid increase in fluorescence within 4 h after induction.

![Arabinose-Inducible Suicide System (pBAD-MazF) Diagram](https://static.igem.wiki/teams/5964/results/img13.webp)

Figure 13. Fluorescent protein expression in engineered strains under different arabinose concentrations.

### Conclusion:

The pBAD promoter exhibits excellent characteristics of low leakage, high inducibility, and dose-dependent response, making it suitable for constructing controllable gene expression systems, and provides a reliable promoter element for subsequent construction of the pBAD-MazF suicide system.

## Arabinose-Inducible Suicide System

### Objective:

Based on validation of pBAD functionality, the toxin gene MazF was substituted for mRFP to construct the pBAD-MazF system, enabling programmed cell death upon arabinose induction for controllable clearance of engineered bacteria within the host.

### Methods:

The MazF gene was placed downstream of the pBAD promoter to construct the recombinant plasmid pSB1A3-pBAD-MazF, which was transformed into *E. coli* DH5α to obtain the engineered strain. The strain was inoculated in LB medium containing 50 μg/mL ampicillin and cultured at 37 °C and 180 rpm until OD₆₀₀ ≈ 0.6. The experimental group was induced with 0.2% L-arabinose to express MazF, while the control group received no inducer. OD₆₀₀ was measured every 2 h for 24 h.

![Arabinose-Inducible Suicide System Diagram](https://static.igem.wiki/teams/5964/results/img14.webp)

Figure 14. Arabinose-inducible suicide system. Figure A: plasmid map; Figure B: gene circuit diagram; Figure C, D: agarose gel electrophoresis of araC-pBAD, MazF.

### Results:

Without induction, the growth curve of the engineered strain was almost identical to that of wild-type DH5α, indicating no leakage expression of MazF and a "shut-off" state of the system. After addition of 0.2% arabinose, OD₆₀₀ rapidly decreased to near zero (0.19 ± 0.16) within 12 h, whereas the control group continued to grow to OD₆₀₀ > 2.31 ± 0.22, showing a significant difference.

![Arabinose-Inducible Suicide System Diagram](https://static.igem.wiki/teams/5964/results/img15.webp)

Figure 15. Arabinose-inducible suicide system. Figure A: growth of engineered and control strains without arabinose; Figure B: growth of engineered strain under different arabinose concentrations.

### Conclusion:

The pBAD-MazF system can efficiently initiate programmed cell death upon arabinose induction, enabling rapid and controllable clearance of engineered bacteria. This provides a "user-triggered" safety switch for clinical intervention, significantly enhancing the biosafety of the system.

## Cold-Inducible Suicide System (pCspA-MazF)

### Functional Validation of pCspA Promoter

### Objective:

To evaluate the activity of the cold-shock protein promoter pCspA at different temperatures, a pCspA-mRFP fluorescent reporter system was constructed to verify its specific activation at low temperatures, providing a basis for constructing an environment-responsive suicide system.

### Methods:

The mRFP gene was placed downstream of the pCspA promoter to construct the recombinant plasmid pSB1A3-pCspA-mRFP, which was transformed into *E. coli* DH5α to obtain the engineered strain. The strain was inoculated in LB medium containing 50 μg/mL ampicillin and cultured at 16 °C, 25 °C, and 37 °C at 180 rpm for 12 h. Samples were collected periodically to measure OD₆₀₀ and mRFP fluorescence intensity, and normalized fluorescence was calculated.

![Cold-Inducible Suicide System (pCspA-MazF) Diagram](https://static.igem.wiki/teams/5964/results/img16.webp)

Figure 16. Temperature-responsive biosensor. Figure A: plasmid map; Figure B: agarose gel electrophoresis of pCspA-mRFP; Figure C: gene circuit diagram.

### Results:

At 16 °C, normalized fluorescence reached 270.84 ± 19.36 AU at 12 h, significantly higher than at 25 °C (240.04 ± 14.51 AU) and 37 °C (67.92 ± 22.24 AU) (*p* < 0.01). Fluorescence remained low at 37 °C, indicating minimal activity of pCspA under body temperature conditions.

![Cold-Inducible Suicide System (pCspA-MazF) Diagram](https://static.igem.wiki/teams/5964/results/img17.webp)

Figure 17. Fluorescent protein expression in temperature-responsive biosensor strains under different temperatures.

### Conclusion:

The pCspA promoter exhibits the ideal characteristic of "activated at low temperature, shut off at body temperature," making it suitable for constructing environment-responsive gene expression systems. It effectively prevents unintended activation of engineered bacteria within the host, ensuring functional specificity.

## Cold-Inducible Suicide System

### Objective:

Based on validation of pCspA functionality, the mRFP gene was replaced with the toxin gene MazF to construct the pCspA-MazF system, enabling automatic clearance of engineered bacteria in cold external environments to prevent ecological spread.

### Methods:

The recombinant plasmid pSB1A3-pCspA-MazF was constructed and transformed into *E. coli* DH5α. The experimental group was cultured at 16 °C and 180 rpm, while the control group was cultured at 37 °C. OD₆₀₀ was measured every 2 h for 24 h. The control group consisted of wild-type DH5α.

![Cold-Inducible Suicide System Diagram](https://static.igem.wiki/teams/5964/results/img18.webp)

Figure 18. Cold-inducible suicide system. Figure A: plasmid map; Figure B: agarose gel electrophoresis of MazF; Figure C: gene circuit diagram.

### Results:

At 16 °C, the engineered strain’s OD₆₀₀ reached only 0.34 ± 0.08 at 8 h, significantly lower than the wild-type control (0.83 ± 0.06, *p* < 0.01), with no further significant growth. At 37 °C, the engineered strain grew normally, showing no difference from the wild type, indicating activation only under low temperature. Additionally, when the constructed in vitro suicide system strain was cultured with wild-type strain at 16 °C, results showed that OD₆₀₀ remained nearly unchanged within 8 h, while the control grew normally. This demonstrates that the suicide system can effectively induce cell death under low temperature.

![Cold-Inducible Suicide System Diagram](https://static.igem.wiki/teams/5964/results/img19.webp)

Figure 19. Cold-inducible suicide system. Figure A: growth of cold-inducible suicide engineered strain after 8 h at different temperatures; Figure B: comparison of growth curves between cold-inducible suicide engineered strain and wild-type strain at 16 °C.

### Conclusion:

The pCspA-MazF system can automatically initiate MazF expression under low temperature, leading to growth arrest and cell death, achieving an "environmental self-destruction" function. As a second safety barrier, this system effectively prevents survival and spread of engineered bacteria after excretion from the host, establishing an "environment-triggered" biocontainment mechanism.

# Summary

This study successfully constructed and validated:

* An engineered bacterial system capable of efficient neohesperidin synthesis, achieving multi-pathway production from hesperetin (1.17 mg/mL) and naringenin (4.55 mg/L) as precursors;
* A pH-responsive targeting synthesis mechanism based on the acidity of the tumor microenvironment, achieving neohesperidin production of 4.20 mg/mL at pH 5.8, significantly higher than under neutral conditions;
* A dual biosecurity protection system combining an "in vivo controllable clearance" suicide system (pBAD-MazF) and an "environmental self-destruction" suicide system (pCspA-MazF), providing a two-layer safety framework with user-triggered and environment-triggered mechanisms, respectively.

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