iGEM 2025 — Team jiashu-china

{% 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

* + [HMB synthesis system](#section-2)
    - [Serotonin synthesis system](#section-3)
      * [Safety system](#section-4)

![Picture Diagram](https://static.igem.wiki/teams/5983/notebook/img1.webp)

# HMB synthesis system

| Date | Experiment | Details | Notes |
| --- | --- | --- | --- |
| 6/29 | Gene Sequence Retrieval & Codon Optimization | Retrieve sequences of AtoB, MvaS, AibAB, LiuC from NCBI. Perform codon optimization for E. coli and remove EcoRI, XbaI, SpeI, PstI sites. | Initial sequences prepared, ready for synthesis. |
| 6/30 | Gene Synthesis Ordering | Submit optimized sequences to Generalbial (China) for synthesis. | Delivery expected in ~5-7 days. |
| 7/1 | PCR Primer Design | Design primers for PCR amplification of synthesized genes; check Tm, GC content, and secondary structures. | Ensure primers compatible with pSB1A3 cloning. |
| 7/2 | Plasmid Backbone Preparation | Linearize pSB1A3 vector; confirm by gel electrophoresis. | Vector ready for cloning. |
| 7/3 | PCR Amplification of Genes | Amplify AtoB, MvaS, AibAB, LiuC fragments from synthesized DNA. | Gel electrophoresis to verify fragment size. |
| 7/4 | PCR Fragment Verification | Run agarose gel; check for correct size and yield. | Some fragments show faint bands, may require re-amplification. |
| 7/5 | Fragment Re-amplification | Re-amplify low-yield fragments; optimize annealing temperature. | Success: clear bands obtained. |
| 7/6 | One-Step Seamless Cloning | Clone PCR fragments into pSB1A3 vector using Seamless Cloning Kit. | Prepare multiple constructs for backup. |
| 7/7 | Transformation into BL21 (DE3) | Transform recombinant plasmids into competent E. coli BL21 cells; plate on LB/Amp. | Colonies appear next day. |
| 7/8 | Colony Screening | Pick single colonies; inoculate in LB/Amp and perform colony PCR to confirm insert presence. | Some colonies fail to amplify; pick additional colonies. |
| 7/9 | Colony PCR Verification | Confirm positive colonies; prepare glycerol stocks for confirmed clones. | Positive clones saved; some clones fail, backup clones maintained. |
| 7/10 | Pre-culture for HMB Production | Inoculate confirmed clones into 5 mL LB/Amp; grow to OD₆₀₀ ~0.6–0.8. | Cultures ready for HMB assay. |
| 7/11 | HPLC Method Setup | Determine λmax for HMB by full-wavelength scan (200–400 nm). | Detection wavelength determined: 208 nm. |
| 7/12 | HPLC Standard Curve | Prepare HMB standard solutions (0.05–2 mg/L) and measure peaks. | Standard curve linear; r² > 0.99. |
| 7/13 | Small-Scale HMB Fermentation | Inoculate clones into M9 medium; 12 h cultivation at 37°C. | OD and supernatant collected for HMB quantification. |
| 7/14 | HMB Quantification | Measure HMB in supernatants via HPLC. | Yield ~60–65 mg/L. |
| 7/15 | Growth Curve Analysis | Monitor OD₆₀₀ of wild-type vs engineered strains. | No significant growth inhibition observed. |
| 7/16 | Glucose Consumption & HMB Correlation | Measure glucose and HMB every 6 h; perform correlation analysis. | Positive correlation observed. |
| 7/17 | Thioesterase Gene PCR Verification | PCR amplify tesB, YciA, MenI; check sequences on gel. | Some fragments faint; re-amplification required. |
| 7/18 | Re-amplification of Thioesterases | Optimize PCR for low-yield genes. | All fragments verified successfully. |
| 7/19 | Thioesterase Cloning into HMB Strain | Clone tesB, YciA, MenI into HMB-producing plasmids. | Prepare constructs for transformation. |
| 7/20 | Transformation & Colony Screening | Transform into BL21-HMB; select colonies and confirm inserts. | YciA clone successful; tesB low efficiency, repeat next day. |
| 7/21 | Re-transformation of tesB | Retry transformation of tesB construct. | Colonies obtained. |
| 7/22 | Small-Scale Fermentation with Thioesterases | Inoculate tesB, YciA, MenI strains into M9 medium; 12 h cultivation. | OD monitored; no growth defects. |
| 7/23 | HMB Quantification with Thioesterases | Measure HMB via HPLC. | YciA: 185 mg/L; TesB: 148 mg/L; MenI: 101 mg/L. |
| 7/24 | Growth & Glucose Analysis | Compare OD and glucose consumption across thioesterase strains. | All strains show normal growth; YciA shows highest carbon utilization efficiency. |
| 7/25 | Replicate Fermentation | Repeat 12 h fermentation in triplicate to ensure reproducibility. | Results consistent; minor variations in yield. |
| 7/26 | Data Analysis & Graphing | Plot HMB production, growth curves, glucose correlation. | Figures prepared for report. |
| 7/27 | Troubleshooting Low-Yield Strains | Investigate MenI low yield; consider promoter strength or codon usage. | Decide to keep for comparison. |
| 7/28 | Final Verification | Repeat key experiments for confirmation. | Data confirmed reproducible. |
| 7/29 | Summary of HMB Expression | Compile HMB yields, growth, carbon flux, and thioesterase effects. | All data documented. |
| 7/30 | Report & Figures Finalization | Prepare experimental report and figures for HMB Expression Verification. | Ready for submission or next experimental phase. |

# Serotonin synthesis system

| Date | Experiment | Details | Notes |
| --- | --- | --- | --- |
| 7/31 | Serotonin gene acquisition and codon optimization | TDC (BBa\_25P80OSX) and TPH (BBa\_25G48CDM) sequences were obtained from NCBI and codon-optimized for E. coli. Restriction sites EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI were removed to comply with RFC#10 standard. | Gene sequences prepared for synthesis. |
| 8/1 | Gene synthesis order | The optimized TDC and TPH genes were submitted to Generalbial (China) for synthesis. | Expected delivery 5–7 days. |
| 8/2 | Primer design | Primers were designed for PCR amplification and cloning. | Tm, GC content, and secondary structure checked. |
| 8/3 | Vector preparation | pET28a(m) vector was prepared and NdeI/XhoI restriction sites verified. | Vector ready for cloning. |
| 8/4 | PCR amplification | TDC and TPH fragments amplified, checked by gel electrophoresis. | Clear bands, sufficient yield. |
| 8/5 | Fragment cloning | TDC and TPH fragments cloned into pET28a(m) using NdeI/XhoI sites. | Multiple construct backups prepared. |
| 8/6 | Transformation into DH5α | Recombinant plasmids transformed into DH5α and plated on LB/Kana plates to select positive colonies. | Some colonies grew slowly; extended incubation needed. |
| 8/7 | Colony screening | Single colonies inoculated into LB/Kana liquid medium and sequenced for verification. | Sequencing confirmed positive clones. |
| 8/8 | Transformation into BL21 | Verified recombinant plasmids transformed into BL21 for expression experiments. | Overnight culture obtained colonies. |
| 8/9 | Small-scale preculture | Positive BL21 strains inoculated into 5 mL LB/Kana, cultured to OD₆₀₀ ~0.6. | Ready for induction. |
| 8/10 | Serotonin induction expression | Inoculated into 20 mL modified M9 medium; at OD₆₀₀ = 0.6, 0.5 mM IPTG added, incubated for 20 h. | Sampling scheduled. |
| 8/11 | Sampling and filtration | 1 mL samples collected every 5 h, filtered through 0.22 μm PES membrane, stored at -20°C. | Samples preserved. |
| 8/12 | Serotonin ELISA measurement | Serotonin concentration measured using 5-HT ELISA kit. | Preliminary titer 1.85 mg/L. |
| 8/13 | Functional verification | Compared serotonin production of wild-type, TDC-only, and TDC+TPH strains. | TDC-only strain produced no detectable serotonin. |
| 8/14 | Data recording and analysis | Serotonin titer over time was recorded and plotted. | Used for yield and expression efficiency analysis. |
| 8/15 | Screening TDC from different sources | Tested TDC genes from rice, Catharanthus, and pig to evaluate effect on serotonin yield. | Catharanthus-derived TDC performed best. |
| 8/16 | Replicate experiments with different TDC | Repeated serotonin production experiment with Catharanthus TDC to verify reproducibility. | Maximum titer reached 2.62 mg/L. |
| 8/17 | Small-scale fermentation repeats | Best TDC strain fermented in 20 mL modified M9 medium three times. | Production stable. |
| 8/18 | OD and growth curve analysis | Compared OD₆₀₀ of different strains to check for growth impact. | All strains grew normally, no significant inhibition. |
| 8/19 | Sample collection and HPLC/ELISA verification | Samples collected again for serotonin measurement to ensure ELISA consistency. | Data consistent. |
| 8/20 | Data analysis and graphing | Plotted serotonin production curves and TDC source comparison graphs. | Figures prepared. |
| 8/21 | Troubleshooting | Checked low-yield or low-expression strains and recorded anomalies. | Some TDC sources performed poorly, kept as controls. |
| 8/22 | Serotonin yield repeat verification | Repeated key experiments to confirm 2.62 mg/L titer. | Results reproducible. |
| 8/23 | System summary | Summarized serotonin synthesis system results, including strain construction, yield, and TDC source comparison. | Data ready for next-stage experiments or reports. |
| 8/24 | Report preparation | Completed serotonin system experimental report and figure compilation. | Prepared for transition to safety system or next project phase. |
| 8/25 | Data review | Final review of experimental data to ensure accuracy and reproducibility. | Experiment completed, data complete. |

# Safety system

| Date | Experiment | Details | Notes |
| --- | --- | --- | --- |
| 9/2 | Safety system design | Designed a controllable safety system using arabinose-inducible promoter PBAD (BBa\_K808000) to drive expression of E. coli toxin MazF for self-lysis. | Genetic circuit drafted. |
| 9/3 | Promoter test setup | mRFP used as reporter to test arabinose-inducible function of PBAD. | Fluorescence/OD₆₀₀ monitored. |
| 9/4 | Promoter function verification | Tested various arabinose concentrations; Fluorescence/OD₆₀₀ increased with arabinose concentration. | PBAD inducible function confirmed. |
| 9/5 | MazF gene synthesis | E. coli MazF gene codon-optimized to meet RFC#10 standard. | Ready for cloning. |
| 9/6 | Construction of PBAD-MazF cassette | Overlap PCR used to place MazF downstream of PBAD, forming PBAD-MazF expression cassette. | Construct verified by gel electrophoresis. |
| 9/7 | Cloning into plasmid | PBAD-MazF cassette cloned into pSB1A3 via XbaI/SpeI restriction sites. | Plasmid ready for transformation. |
| 9/8 | Transformation into DH5α | Recombinant plasmid transformed into E. coli DH5α; plated on LB/Ampicillin plates. | Positive colonies obtained. |
| 9/9 | Preculture of engineered strain | DH5α-PBAD-MazF and wild-type DH5α inoculated into 5 mL LB + 100 μg/mL Ampicillin, grown overnight at 37°C, 150 rpm. | Activation culture ready. |
| 9/10 | Induction setup | Overnight cultures 1:100 inoculated into 20 mL fresh LB, initial OD₆₀₀ = 0.1; induced at OD₆₀₀ ≈ 0.4 with 2% L-arabinose. | Induction for growth curve assay. |
| 9/11 | Growth curve monitoring | Cell growth monitored using FlexStation 3 microplate reader to generate growth curves. | Observed DH5α-PBAD-MazF growth inhibition under arabinose induction. |
| 9/12 | System effect verification | Compared growth of induced, non-induced, and wild-type controls. | Only induced DH5α-PBAD-MazF showed growth suppression. |
| 9/13 | Data collection and analysis | Collected OD₆₀₀ readings, plotted growth curves. | Verified controllable “suicide switch” function. |
| 9/14 | Repeat induction experiment | Repeated arabinose induction to confirm reproducibility. | System function consistent. |
| 9/15 | Troubleshooting | Checked any unexpected growth in engineered strains; ensured PBAD-MazF properly activated. | Minor adjustments made; system reliable. |
| 9/16 | Summary and report | Summarized results, including strain construction, PBAD inducibility, MazF activation, and growth inhibition. | Safety system verified and ready for integration with metabolic experiments. |

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{% endblock %}

{% 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 %}
{% block page\_content %}

Content

* [Overview](#section-1)
  + [HMB Synthesis System](#section-2)
    - [1.1 Construction of the HMB Synthesis System](#subsection-3)
    - [1.2 Introduction of Thioesterases to Enhance HMB Production](#subsection-4)
  + [Serotonin Biosynthesis System](#section-5)
    - [2.1 Construction of Serotonin Biosynthesis System](#subsection-6)
    - [2.2 Screening TDC to Improve Serotonin Production](#subsection-7)
  + [Safety System](#section-8)
    - [3.1 Arabinose Promoter Testing](#subsection-9)
    - [3.2 Suicide System Validation](#subsection-10)
  + [Summary](#section-11)
    - [Reference](#section-12)

# Overview

This study aimed to engineer Escherichia coli to construct and optimize production systems for two bioactive molecules: HMB (β-hydroxy-β-methylbutyrate) and serotonin (5-hydroxytryptamine, 5-HT). HMB, an important muscle-protective agent, is primarily synthesized via the leucine metabolic pathway. We designed and constructed an engineered strain containing the key genes for HMB synthesis. To enhance HMB production, thioesterases were introduced to alleviate metabolic bottlenecks.

Serotonin, a crucial neurotransmitter, is widely involved in the regulation of mood, sleep, and other physiological processes. We further designed and constructed a serotonin synthesis system, optimizing enzyme expression to increase serotonin yield. Finally, a safety system was implemented to prevent potential biosafety issues associated with the engineered strains.

![Overview Diagram](https://static.igem.wiki/teams/5983/engineering/img1.webp)

# HMB Synthesis System

## 1.1 Construction of the HMB Synthesis System

### Design

HMB (β-hydroxy-β-methylbutyrate) is a bioactive molecule derived from leucine metabolism and has been widely applied in the prevention and mitigation of muscle atrophy. HMB exhibits significant anti-proteolytic and protein synthesis-promoting activities, making it an effective muscle-protective agent [1].

Escherichia coli (E.coli) was selected as the microbial chassis for HMB production due to its multiple advantages, including rapid growth, simple cultivation, ease of genetic manipulation, low cost, and precise genome editing capabilities, making it an ideal host for the production of proteins and other bioactive molecules [2].

The de novo HMB synthesis pathway (3-hydroxy-3-methylbutyrate) consists of five key enzymes: AtoB (acetoacetyl-CoA thiolase), MvaS (HMG-CoA synthase), AibAB (3-methylglutaconyl-CoA decarboxylase), LiuC (3-methylglutaconyl-CoA hydratase), and YciA (thioesterase). This pathway uses three molecules of acetyl-CoA as substrates to produce one molecule of HMB, demonstrating high theoretical carbon conversion efficiency and being independent of ATP or NADH.

![1.1 Construction of the HMB Synthesis System Diagram](https://static.igem.wiki/teams/5983/engineering/img2.webp)

Figure 1: De Novo Synthesis Method of HMB

### Bulid

To obtain a strain capable of efficient de novo HMB synthesis, we designed a gene circuit containing the key genes for HMB biosynthesis. E. coli BL21 was chosen as the microbial chassis for constructing the engineered strain. Using genetic engineering techniques, we cloned acetoacetyl-CoA thiolase (AtoB) from E. coli, 3-hydroxy-3-methylglutaryl-CoA synthase (MvaS) from Staphylococcus aureus, glutaconyl-CoA decarboxylase (AibAB) from Bacillus subtilis, and 3-methylglutaconyl-CoA hydratase (LiuC) into the pET28A(m) plasmid, which was subsequently transformed into E. coli BL21 (Figure 2).

![1.1 Construction of the HMB Synthesis System Diagram](https://static.igem.wiki/teams/5983/engineering/img3.webp)

Figure 2 Construction of HMB-Producing Strain

We first obtained the AtoB, MvaS, AibAB, and LiuC genes from NCBI and performed codon optimization based on E. coli codon preference, while removing EcoRI, XbaI, SpeI, and PstI restriction sites (sequence verification was conducted using SnapGene®) before outsourcing synthesis. The target fragments were then PCR-amplified and verified by agarose gel electrophoresis (Figure 3A). Subsequently, the fragments were cloned into the pSB1A3 vector (BioBricks™ standard RFC#10) using one-step seamless cloning (Seamless Cloning Kit, D7010, Beyotime).

The resulting recombinant plasmids were transformed into E. coli BL21 (DE3) competent cells and plated on LB agar containing 100 μg/mL ampicillin (Amp⁺, pH 7.0). After incubation at 37°C for 16 h, single colonies were obtained (Figure 3B). Single colonies were inoculated into 5 mL LB/Amp⁺ liquid medium and cultured at 37°C, 220 rpm until OD₆₀₀ reached 0.6–0.8. Positive clones were further verified by colony PCR (2×Taq Master Mix, Vazyme) (Figure 3B).

![1.1 Construction of the HMB Synthesis System Diagram](https://static.igem.wiki/teams/5983/engineering/img4.webp)

Figure 3 Verification of HMB-Related Fragments and Single Colony Screening

### Test

To determine the maximum absorption wavelength (λmax) of the target compound, a full-wavelength scan was performed using an Agilent 1200 series HPLC system equipped with a diode array detector (DAD) under the previously described chromatographic conditions. The detector scan range was set from 200 to 400 nm, with a bandwidth of 4 nm and a sampling frequency of 1 Hz, continuously acquiring spectral data throughout the retention time. The UV spectrum at the peak apex of the target compound was extracted to obtain its characteristic absorption curve, which was then used to determine the λmax, selecting an appropriate wavelength for quantitative detection. The experimentally determined detection wavelength for HMB was 208 nm (Figure 4).

![1.1 Construction of the HMB Synthesis System Diagram](https://static.igem.wiki/teams/5983/engineering/img5.webp)

Figure 4 Determination of HMB Detection Wavelength

The HMB content was analyzed using an Agilent 1200 series HPLC system equipped with a C18 reverse-phase column (250 mm × 4.6 mm, 5 μm). The column temperature was maintained at 30°C. The mobile phase consisted of eluent A (0.02 M KH2PO4, pH 3.0) and eluent B (acetonitrile). The elution gradient was set as follows: initial 95% A; linearly decreased to 40% A from 8 to 8.5 min; maintained at 40% A from 8.5 to 13.0 min; linearly returned to 95% A from 13.0 to 13.5 min; maintained at 95% A from 13.5 to 22.0 min. The flow rate was 1.0 mL/min, and the injection volume was 10 μL. The detection wavelength was set at 208 nm (Table 1).

A series of HMB standard solutions (0.05–2.00 mg/L) were measured at 208 nm to generate a standard curve, which was subsequently used to calculate the HMB concentration in the samples (Figure 5) [1].

Table 1. HPLC Program for HMB Detection

|
|  |

Time (min) |

Eluent A |

Eluent B |

Notes |

|
|  |

0–8.0 |

95% |

5% |

Initial conditions |

|
|  |

8.0–8.5 |

95%→40% |

5%→60% |

Linear gradient |

|
|  |

8.5–13.0 |

40% |

60% |

Hold at constant composition |

|
|  |

13.0–13.5 |

40%→95% |

60%→5% |

Linear return to initial conditions |

|
|  |

13.5–22.0 |

95% |

5% |

Hold at initial conditions until the end of analysis |

![1.1 Construction of the HMB Synthesis System Diagram](https://static.igem.wiki/teams/5983/engineering/img6.webp)

Figure 5 HMB Standard Peaks at 208 nm

To determine the HMB production of the engineered strain in M9 medium after 12 hours of cultivation, the strains were first activated in LB/Amp⁺ medium and then inoculated into M9 medium. The cell growth was monitored in real-time by measuring OD₆₀₀. Samples were collected at different time points, centrifuged, and the supernatants were used for HMB quantification. Each experiment was performed in triplicate, and comparisons between groups were analyzed using the Student’s t-test, with p < 0.05 considered statistically significant.

The results showed that the engineered E. coli produced 65.13 mg/L HMB after 12 hours of cultivation at 37°C, whereas the wild-type BL21 did not produce detectable HMB (Figure 6).

![1.1 Construction of the HMB Synthesis System Diagram](https://static.igem.wiki/teams/5983/engineering/img7.webp)

Figure 6 HMB Production Testing

To evaluate whether HMB production affects the growth of the engineered strain, the OD₆₀₀ of both wild-type and engineered E. coli was measured every 6 hours over a 24-hour cultivation period. The results demonstrated that HMB production did not significantly affect the growth of the engineered strain (Figure 7).

![1.1 Construction of the HMB Synthesis System Diagram](https://static.igem.wiki/teams/5983/engineering/img8.webp)

Figure 7 HMB-Producing Strain Growth Curve

To quantify the carbon source utilization efficiency and metabolic pathway effectiveness of the engineered strain, the glucose consumption and HMB production were dynamically monitored every 6 hours over a 24-hour cultivation period, followed by correlation analysis. The results showed a significant positive correlation between glucose consumption and HMB production (Figure 8).

This observation directly demonstrates that glucose as a carbon source is efficiently channeled into the de novo HMB biosynthesis pathway, rather than being redundantly consumed in side metabolism or for non-productive biomass growth, thereby confirming the functional integrity and metabolic rationality of the engineered pathway.

Importantly, these results provide essential experimental data for the subsequent calculation of carbon flux efficiency and carbon source utilization rate. Efficient carbon allocation is not only crucial for increasing HMB yield but also aligns with the requirements of resource-limited scenarios, such as space stations or closed ecosystems, where low-consumption, high-efficiency biosynthetic systems are essential. Overall, the positive correlation between glucose consumption and HMB generation over 24 hours confirms the engineered strain’s capacity for de novo HMB biosynthesis (Figure 8).

![1.1 Construction of the HMB Synthesis System Diagram](https://static.igem.wiki/teams/5983/engineering/img9.webp)

Figure 8 HMB Production Time-Course Curve

In this study, we successfully constructed an engineered strain capable of HMB biosynthesis. Using the optimized HPLC method, results showed that the engineered strain produced approximately 65 mg/L HMB after 12 hours of cultivation in M9 medium, and HMB production had no significant impact on host growth. Further analysis revealed a positive correlation between HMB production and glucose consumption, demonstrating that the engineered strain can efficiently convert carbon sources into the target product.

### Learn

Although we successfully established the HMB-producing strain, the initial HMB yield of 65 mg/L was insufficient to effectively counteract muscle atrophy. Therefore, through discussions with researchers and literature review, we decided to screen thioesterases from different sources to enhance HMB production.

## 1.2 Introduction of Thioesterases to Enhance HMB Production

### Design

The core rationale for introducing thioesterases to boost HMB production is their ability to efficiently hydrolyze HMB-CoA, releasing free HMB. This alleviates metabolic flux bottlenecks, reduces feedback inhibition, and improves both accumulation and secretion of HMB, thereby increasing the overall yield [3]. Therefore, we heterologously expressed thioesterases in plasmids to evaluate their effects on HMB production.

### Build

First, we obtained thioesterase genes tesB, YciA, and MenI from E. coli via NCBI. Each gene was then cloned into plasmid 2 for expression (Figure 9).

![1.2 Introduction of Thioesterases to Enhance HMB Production Diagram](https://static.igem.wiki/teams/5983/engineering/img10-new.webp)

Figure 9 Thioesterase-Related Plasmid Construction

### Test

First, we successfully amplified the thioesterase genes tesB, YciA, and MenI (Figure 10).

![1.2 Introduction of Thioesterases to Enhance HMB Production Diagram](https://static.igem.wiki/teams/5983/engineering/img11.webp)

Figure 10 Thioesterase Expression Verification by Agarose Gel Electrophoresis

Subsequently, HMB production was tested using the same method. The results showed that: tesB expression led to an HMB titer of 148.32 mg/L after 12 hours. YciA expression exhibited the best performance, producing 185.86 mg/L HMB after 12 hours. MenI expression resulted in the lowest titer among the three, 101.29 mg/L, but still higher than the strain without thioesterase (Figure 11).

![1.2 Introduction of Thioesterases to Enhance HMB Production Diagram](https://static.igem.wiki/teams/5983/engineering/img12.webp)

Figure 11 HMB Production under Different Thioesterases

### Learn

We successfully screened the thioesterase YciA, which could increase HMB production to 185.86 mg/L within 12 hours. Although the constructed HMB biosynthesis system mainly aims to alleviate muscle loss through the de novo synthesis pathway, the health challenges faced by humans in space environments go far beyond muscle atrophy. Besides metabolic regulation, the balance of neurotransmitter systems is equally crucial. Therefore, we decided to design a serotonin biosynthesis system to counteract neural and emotional dysregulation caused by the space environment.

# Serotonin Biosynthesis System

## 2.1 Construction of Serotonin Biosynthesis System

### Design

Serotonin (5-hydroxytryptamine, 5-HT) is an important monoamine neurotransmitter that participates extensively in human signal transduction and is considered a key factor in regulating mood, sleep, appetite, cognition, and gut function. Similarly, we selected Escherichia coli as the microbial chassis to test serotonin production.

### Build

Serotonin biosynthesis primarily relies on tryptophan (TRP) and is catalyzed sequentially by two key enzymes: first, tryptophan hydroxylase (TPH) converts TRP into the intermediate 5-hydroxytryptophan (5-HTP); then, tryptophan decarboxylase (TDC) decarboxylates 5-HTP to produce the active neurotransmitter 5-HT.

We first constructed the serotonin biosynthesis system by obtaining TDC and TPH genes from NCBI (synthesized by Generalbial, China) and performed codon optimization for E. coli, while removing EcoRI, XbaI, SpeI, PstI, NdeI, XhoI restriction sites to comply with the RFC#10 standard and pET28a(m) cloning compatibility. The genes were then cloned into the pET28a(m) vector using NdeI and XhoI restriction sites. The recombinant plasmids were transformed into E. coli DH5α and BL21, and positive clones were screened on LB agar plates containing 100 μg/mL kanamycin (Kana, with 1.5% agar) and verified by sequencing (Beijing, Qingke), yielding the recombinant engineered strains (Figure 12).

![2.1 Construction of Serotonin Biosynthesis System Diagram](https://static.igem.wiki/teams/5983/engineering/img13.webp)

Figure 12 Construction of Serotonin-Producing Strains

(A) Serotonin Biosynthesis Pathway Construction (B) Serotonin Biosynthesis Plasmid Map (C) Agarose Gel Electrophoresis Verification of Serotonin-Related Enzyme Amplification

### Test

To measure serotonin (5-HT) content, the engineered E. coli strain was inoculated at a 1:100 ratio into 20 mL of modified M9 medium. When the culture reached OD600 = 0.6, 0.5 mM IPTG was added to induce expression, and the culture was incubated for an additional 20 hours. 1 mL of fermentation broth was filtered through a 0.22 μm hydrophilic PES membrane (BS-PES-45, Biosharp), and samples were collected every 5 hours and stored at -20°C. The serotonin concentration in the culture medium was then analyzed using a 5-HT ELISA kit (JL53170, Jonln). Serotonin levels were calculated based on a standard curve. The results showed that the engineered E. coli produced 1.85 mg/L serotonin, whereas the wild-type strain and E. coli expressing only TDC did not produce detectable serotonin (Figure 13).

![2.1 Construction of Serotonin Biosynthesis System Diagram](https://static.igem.wiki/teams/5983/engineering/img14.webp)

Figure 13 Serotonin Production Assay

(A) Serotonin production measured every 6 hours over 24 hours.

(B) Functional verification of enzymes involved in serotonin biosynthesis.

### Learn

We successfully constructed a serotonin biosynthesis system, achieving a production level of 1.85 mg/L. However, for gut microbiota applications, higher product concentrations are necessary to ensure effective secretion and functional impact. Therefore, we screened TDC enzymes from different sources to further improve serotonin yield.

## 2.2 Screening TDC to Improve Serotonin Production

### Design

By searching databases and literature, we selected TDC enzymes from rice, catharanthus, and pig to compare their serotonin production capabilities.

### Build

The TDC genes from catharanthus and pig were cloned into plasmids for expression in E. coli (Figure 14).

![2.2 Screening TDC to Improve Serotonin Production Diagram](https://static.igem.wiki/teams/5983/engineering/img15-new.webp)

Figure 15 TDC Amplification Verification

### Test

We successfully amplified the TDC genes from catharanthus and pig (Figure 15 A, B).

![2.2 Screening TDC to Improve Serotonin Production Diagram](https://static.igem.wiki/teams/5983/engineering/img16.webp)

Figure 15 TDC Source Screening

We then compared the serotonin production of TDCs from different sources. The results showed that: Rice-derived TDC produced 1.85 mg/L serotonin; Catharanthus-derived TDC had the best performance, producing 2.62 mg/L serotonin; Pig-derived TDC produced only 0.28 mg/L serotonin (Figure 15 C).

### Learn

By screening TDCs from different sources, we successfully increased serotonin production to 2.62 mg/L. In the future, we plan to screen additional TDC sources to further enhance serotonin yield to meet our target.

# Safety System

## 3.1 Arabinose Promoter Testing

### Design

To ensure that engineered genetic constructs do not escape into the environment and can be cleared from the host when needed, we used an arabinose-inducible promoter as a controllable switch to trigger bacterial cell death. The team first validated the function of the existing part BBa\_K808000 in *E. coli* DH5α.

### Build

To verify that the arabinose promoter functions properly in *E. coli* DH5α, we fused it with an mRFP fragment. The promoter activity was then evaluated by measuring fluorescence at different arabinose concentrations (Figure 16).

![3.1 Arabinose Promoter Testing Diagram](https://static.igem.wiki/teams/5983/engineering/img17-new.webp)

Figure 16. Construction of plasmid for testing arabinose promoter functionality

### Test

We systematically tested the effect of different arabinose concentrations on mRFP expression. The results showed that the fluorescence/OD600 ratio increased significantly with higher arabinose concentrations, confirming that the arabinose promoter can dose-dependently induce target protein expression (Figure 17).

![3.1 Arabinose Promoter Testing Diagram](https://static.igem.wiki/teams/5983/engineering/img18.webp)

Figure 17. Dose-dependent induction of red fluorescent protein expression by different concentrations of arabinose

### Learn

We successfully validated the arabinose-inducible system in *E. coli* DH5α, laying a solid foundation for the construction of the suicide system.

## 3.2 Suicide System Validation

### Design

MazF is the toxin protein from the E. coli *mazEF* toxin-antitoxin system, which specifically cleaves bacterial mRNA at ACA sequences, blocking protein synthesis and causing rapid cell death. Therefore, we chose to use arabinose-inducible expression of MazF to quickly eliminate administered engineered bacteria in the gut, ensuring biosafety.

### Build

MazF expression was placed under the control of the arabinose promoter to allow inducible activation of the suicide system.

![3.2 Suicide System Validation Diagram](https://static.igem.wiki/teams/5983/engineering/img19.webp)

Figure 18. Construction and Validation of the Safety (Suicide) System

(A) Gene circuit diagram of the safety system (B) Plasmid map of the safety system (C) Agarose gel electrophoresis verifying the expression of safety system genes

### Test

The experimental results showed that under L-arabinose induction, the growth of the engineered strain DH5α-PBAD-MazF was significantly inhibited, whereas the uninduced group and wild-type control strain exhibited no noticeable growth changes. This indicates that the PBAD-MazF system can effectively trigger toxin expression under arabinose induction, suppress host cell growth, and validates its feasibility as a controllable “suicide switch” for biosafety control in synthetic biology.

![3.2 Suicide System Validation Diagram](https://static.igem.wiki/teams/5983/engineering/img20.webp)

Figure 19 Suicide System Validation

### Learn

We successfully designed and constructed an L-arabinose-inducible MazF safety system and validated its feasibility in Escherichia coli DH5α.

# Summary

In this study, we successfully established HMB and serotoninsynthesis systems. The success of the HMB system not only provides a potential solution for mitigating muscle loss but also demonstrates the possibility of increasing product yield through genetic engineering. The construction of the serotonin system provides a preliminary approach to address neurological challenges in the space environment. Meanwhile, we designed and validated a safety system. In the future, we will further optimize these systems, increase yield, and explore their practical applications.

# Reference

[1] Holeček M. (2017). Beta-hydroxy-beta-methylbutyrate supplementation and skeletal muscle in healthy and muscle-wasting conditions. Journal of cachexia, sarcopenia and muscle, 8(4), 529–541.

[2] Lee S. Y. (1996). High cell-density culture of Escherichia coli. Trends in biotechnology, 14(3), 98–105.

[3] Huang, S. J., Lai, M. J., Chen, A. Y., & Lan, E. I. (2024). De novo biosynthesis of 3-hydroxy-3-methylbutyrate as anti-catabolic supplement by metabolically engineered Escherichia coli. Metabolic engineering, 84, 48–58.

{% 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

* [Construction of HMB-Producing Engineered Strain](#section-1)
  + [HMB Production Assay](#section-2)
    - [Glucose Consumption Analysis](#section-3)
      * [Heterologous Expression of Thioesterases](#section-4)
        + [Construction of Serotonin-Producing Engineered Strains](#section-5)
          - [Serotonin Production Assay](#section-6)
            * [Construction of Arabinose-Inducible Reporter Strain](#section-7)
              + [Reporter Strain Assay](#section-8)
                - [Construction of Safety System](#section-9)
                  * [Safety System Assay](#section-10)
                    + [Statistical Analysis](#section-11)

# Construction of HMB-Producing Engineered Strain

![Construction of HMB-Producing Engineered Strain Diagram](https://static.igem.wiki/teams/5983/experiments/img1.webp)

Figure 1 Experimental Workflow

Based on the codon preference of Escherichia coli, the HMB synthesis-related genes AtoB, Mvas, LiuC, and AibA were codon-optimized, while EcoRI, XbaI, SpeI, and PstI restriction sites were removed (sequence verification was performed using SnapGene®). The optimized genes were synthesized by Generalbial (China) and cloned into the pSB1A3 vector (BioBricks™ standard RFC#10). A polycistronic expression cassette was constructed using the constitutive promoter J23100 (BBa\_J23100) and ribosome binding site B0034 (BBa\_B0034), and then assembled into the vector via one-step seamless cloning (Seamless Cloning Kit, D7010, Beyotime). The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells and plated on LB agar plates containing 100 μg/mL ampicillin (Amp⁺) (pH 7.0), followed by incubation at 37°C for 16 h. Single colonies were picked and inoculated into 5 mL LB/Amp⁺ liquid medium, incubated at 37°C, 220 rpm until OD₆₀₀ = 0.6–0.8. Positive clones were verified by colony PCR (2×Taq Master Mix, Vazyme). Sequencing verification was performed by Qingke Biotechnology (Beijing, China). The sequencing-correct BL21-HMB strain was inoculated into 5 mL LB/Amp⁺ medium and cultured at 37°C, 200 rpm. The growth curve was monitored to mid-log phase (OD₆₀₀ ≈ 0.6) using FlexStation 3 (Molecular Devices, USA). Finally, 1 mL of bacterial culture was mixed with 1 mL of 50% glycerol and stored at -20°C for future use.

# HMB Production Assay

100 μL of -20°C glycerol stock of the HMB-producing strain was inoculated into 5 mL LB medium containing 100 μg/mL ampicillin (LB/Amp⁺) and cultured at 37°C, 150 rpm overnight to reactivate the strain. The next day, the overnight culture was centrifuged at 8,000×g, 4°C for 10 min to collect the cell pellet. The pellet was washed three times with phosphate-buffered saline (PBS, pH 7.4) and resuspended in 50 mL M9 minimal medium (A510881, Sangon Biotech) supplemented with 20 g/L glucose, 0.1 g/L thiamine hydrochloride (A8084, Innochem), 1 mM magnesium sulfate (MgSO₄), 50 μM calcium chloride (CaCl₂), and 100 μg/mL Amp.

The bacterial growth density was monitored in real time using a FlexStation 3 multifunctional microplate reader (Molecular Devices, USA) at 600 nm (OD₆₀₀). At designated time points, 1 mL of culture was centrifuged at 18,000×g, 4°C for 2 min to collect the supernatant for HMB quantification.

HMB content was analyzed by high-performance liquid chromatography (HPLC), following a previously reported method with slight modifications. The analysis was performed using an Agilent 1200 HPLC system equipped with a C18 reverse-phase column (250 mm × 4.6 mm, 5 μm) at 30°C. The mobile phase consisted of eluent A (0.02 mol/L potassium dihydrogen phosphate, pH 3.0) and eluent B (acetonitrile), with the following gradient: initial 95% A; 8–8.5 min linear transition to 40% A; 8.5–13.0 min hold at 40% A; 13.0–13.5 min linear return to 95% A; 13.5–22.0 min hold at 95% A. The flow rate was 1.0 mL/min, injection volume was 10 μL, and detection wavelength was 208 nm.

A standard curve was generated using HMB standard solutions (0.05–2.00 mg/mL), which was then used to calculate the actual HMB content in the samples.

Table 1. HPLC Program for HMB Detection

|
|  |

Time (min) |

Eluent A |

Eluent B |

Notes |

|
|  |

0–8.0 |

95% |

5% |

Initial conditions |

|
|  |

8.0–8.5 |

95%→40% |

5%→60% |

Linear gradient |

|
|  |

8.5–13.0 |

40% |

60% |

Hold at constant composition |

|
|  |

13.0–13.5 |

40%→95% |

60%→5% |

Linear return to initial conditions |

|
|  |

13.5–22.0 |

95% |

5% |

Hold at initial conditions until the end of analysis |

# Glucose Consumption Analysis

1 mL of fermentation broth was centrifuged at 10,000×g for 5 min to collect the supernatant, which was stored at -20°C. The glucose content of the samples was determined using a Glucose Assay Kit (60408ES60, Yeasen, China). The total glucose consumption was then calculated.

Detection samples were prepared according to Table 2.

Table 2. Sample Loading in 96-Well Plate (Colorimetric Measurement Using Microplate Reader)

|
|  |

Type of Well |

Blank Well |

Standard Well |

Sample Well |

|
|  |

Distilled Water (μL) |

2.5 |

/ |

/ |

|
|  |

Standard Solution (μL) |

/ |

2.5 |

/ |

|
|  |

Sample (μL) |

/ |

/ |

2.5 |

|
|  |

Working Reagent (μL) |

250 |

250 |

250 |

Gently shake the microplate and incubate at 37°C for 10 min. Measure the absorbance of each well at 505 nm using a microplate reader.

# Heterologous Expression of Thioesterases

Based on the codon preference of Escherichia coli, the thioesterase genes tesB, YciA, and MenI were codon-optimized, while restriction sites EcoRI, XbaI, SpeI, and PstI were removed (sequence verification via SnapGene®). The optimized genes were synthesized by Generalbial (China) and cloned into a plasmid. A polycistronic expression cassette was constructed using the constitutive promoter J23100 (BBa\_J23100) and the ribosome binding site B0034 (BBa\_B0034). Plasmid transformation, engineered strain selection, and HMB production testing were performed as described above.

# Construction of Serotonin-Producing Engineered Strains

TDC and TPH genes were synthesized by Generalbial (China) and codon-optimized for E.coli, with removal of EcoRI, XbaI, SpeI, PstI, NdeI, XhoI restriction sites to comply with RFC#10 standard and pET28a(m) cloning compatibility. Genes were cloned into the pET28a(m) vector via NdeI and XhoI restriction sites. E.coli BL21 (DE3) (D1009S) and DH5α (D0351) supercompetent cells were purchased from Beyotime (China). Recombinant plasmids were transformed into DH5α and BL21. Positive clones were selected on LB agar plates containing 100 μg/mL kanamycin (Kana, 1.5% agar) and sequencing-verified (Qingke Biotechnology, Beijing, China). The engineered strains were stored at -80°C with 25% (v/v) glycerol. Cultivation conditions: 37°C, 150 rpm in LB broth containing 100 μg/mL Kana (G3102, Servicebio, China).

# Serotonin Production Assay

The engineered strain was inoculated at a 1:100 ratio into 20 mL modified M9 medium, containing 1× M9 salts (A510881, Sangon Biotech), 0.2% (w/v) glucose, 0.1% (w/v) casein amino acids (C304284, Aladdin), 1 mM MgSO₄, 50 pM FeCl₃, and 50 mg/L L-tryptophan (A64769, Innochem). Kanamycin final concentration: 100 μg/mL. Cultured at 37°C, 180 rpm for 3–5 h until OD₆₀₀ = 0.6, then 0.5 mM IPTG was added and cultivation continued for 20 h.

1 mL of fermentation broth was filtered through a 0.22 μm hydrophilic PES membrane (BS-PES-45, Biosharp), and samples were collected every 5 h, stored at -20°C. Serotonin content was analyzed using a 5-HT ELISA Kit (JL53170, Jonln), and concentration was calculated from a standard curve.

Experimental Procedure:

1. Remove the required microplate strips from room temperature-equilibrated foil bags; store remaining strips at 4°C.
2. Sample addition: Add 50 μL of each sample or standard solution per well; blank wells receive 50 μL universal diluent. Immediately add 50 μL Biotin-antibody working solution per well. Cover the plate and incubate at 37°C for 60 min. *(Recommendation: dilute samples at least 1:1 with universal diluent to reduce matrix effects; multiply by dilution factor when calculating concentrations; replicate wells are recommended.)*
3. Washing: Discard liquid; add 300 μL 1× wash buffer per well; stand 1 min; remove buffer; blot on absorbent paper; repeat 3 times (or use a plate washer).
4. Add enzyme conjugate working solution:100 μL per well, cover, incubate 37°C for 30 min.
5. Wash: Repeat step 3, 5 times.
6. Add substrate:90 μL TMB per well, cover, incubate 37°C in dark for 15 min.
7. Add stop solution:50 μL per well, measure OD at 450 nm immediately.

# Construction of Arabinose-Inducible Reporter Strain

PBAD promoter (K808000), RBS B0034, and red fluorescent protein mRFP were synthesized. mRFP was placed downstream of PBAD-B0034, codon-optimized for E. coli, and EcoRI, XbaI, SpeI, PstI restriction sites removed to meet RFC#10 standard (Genewiz, USA). The PBAD-mRFP cassette was cloned into pSB1A3 vector via XbaI and SpeI sites. Recombinant plasmids were transformed into E. coli DH5α. Positive clones were sequencing-verified (Qingke Biotechnology, Beijing, China). Cultivation: 37°C, LB medium. Bacterial growth was monitored by OD₆₀₀ using a spectrophotometer (GenStar, China).

# Reporter Strain Assay

Inoculate the arabinose-inducible reporter strain at 1:100 into 5 mL LB + 50 μg/mL Amp, add different L-arabinose concentrations, incubate at 37°C, 180 rpm. Collect 200 μL samples at different time points. Measure fluorescence (Ex 584 nm / Em 607 nm) and OD₆₀₀ using Multiskan GO microplate reader. Calculate normalized fluorescence (Fluorescence/OD₆₀₀).

# Construction of Safety System

E. coli toxin protein MazF was synthesized and codon-optimized to meet RFC#10 standard. mazF was placed downstream of PBAD promoter via overlap PCR, then cloned into pSB1A3 vector via XbaI and SpeI. Recombinant plasmids were transformed into E. coli DH5α, screened on LB agar + 100 μg/mL Amp (1.5% agar), and sequencing-verified. DH5α-PBAD-mazF was stored at -20°C with 25% (v/v) glycerol, cultured at 37°C, 180 rpm in LB broth + 100 μg/mL Amp.

# Safety System Assay

Frozen DH5α-PBAD-mazF and wild-type DH5α were activated overnight in 5 mL LB + 100 μg/mL Amp at 37°C, 150 rpm, then inoculated 1:100 into 20 mL LB + Amp (OD₆₀₀ = 0.1). At OD₆₀₀ ≈ 0.4, add 0.2% L-arabinose to induce MazF expression. Monitor growth curves with FlexStation 3 (Molecular Devices, USA).

# Statistical Analysis

Data were analyzed using GraphPad Prism and presented as mean ± SD. Multiple group comparisons: one-way ANOVA with Tukey’s post hoc test. Two-group comparisons: Student’s t-test. p < 0.05 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

* [Overview](#section-1)
  + [HMB Expression Verification](#section-2)
    - [Serotonin Expression Verification](#section-3)
      * [Construction and Verification of the Safety System](#section-4)
        + [Summary](#section-5)
          - [Reference](#section-6)

# Overview

To achieve the production of key metabolites through synthetic biology, we conducted a series of experimental works focusing on HMB synthesis, serotonin synthesis, and safety system design. First, we successfully constructed engineered strains capable of de novo HMB synthesis, and verified their yield and impact on host growth using HPLC. Further, we optimized the HMB production level by employing thioesterase. Second, we constructed a serotonin synthesis system by co-expressing TDC and TPH in Escherichia coli, and confirmed serotonin production and secretion through ELISA, while screening TDCs from different sources to enhance the yield. Finally, we designed and validated an arabinose-inducible suicide system, providing a biosafety safeguard for the application of the engineered strains. Overall, the experimental results demonstrate that we not only successfully achieved the synthesis of the target metabolites, but also conducted systematic explorations in yield optimization and biological safety, laying a solid foundation for future applications.

# HMB Expression Verification

### Strain Construction

![HMB Expression Verification Diagram](https://static.igem.wiki/teams/5983/results/img1.webp)

Figure 1 Construction of HMB-Producing Strains

(A) Plasmid design for HMB synthesis (B) Plasmid map of HMB synthesis construct

To obtain strains capable of efficient de novo HMB synthesis, we designed a gene circuit containing the key genes for HMB production (Figure 1A). E. coli BL21 was chosen as the host chassis to construct the engineered strain, and the following genes were cloned into BL21: acetoacetyl-CoA thiolase (AtoB, BBa\_25VQVUES) from *E. coli*, 3-hydroxymethylglutaryl-CoA synthase (MvaS, BBa\_25PLCDZA) from *Staphylococcus aureus*, glutaconyl-CoA decarboxylase (AibAB, BBa\_2554PQS6 and BBa\_25EFNVLR) from *Corynebacterium glutamicum*, and \*\*3-methylglutaconyl-CoA hydratase (LiuC, BBa\_25P2LT86)\*. The resulting engineered strain was named BL21-HMB.

First, sequences of AtoB, MvaS, AibAB, and LiuC were retrieved from NCBI, and codon optimization was performed according to E. coli codon preference, while removing EcoRI, XbaI, SpeI, and PstI restriction sites (sequence verified using SnapGene®) and synthesized by Generalbial (China). The target fragments were PCR-amplified, and their correct size was confirmed by gel electrophoresis (Figure 2A). The fragments were then cloned into the pSB1A3 vector (BioBricks™ standard RFC#10) using one-step seamless cloning (Seamless Cloning Kit, D7010, Beyotime). The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells, plated on LB agar containing 100 μg/mL ampicillin (Amp⁺, pH 7.0), and incubated at 37°C for 16 h to obtain single colonies (Figure 2B). Single colonies were inoculated into 5 mL LB/Amp⁺ liquid medium, cultured at 37°C, 220 rpm until OD₆₀₀ = 0.6–0.8, and verified by colony PCR (2× Taq Master Mix, Vazyme) to confirm positive clones.

![HMB Expression Verification Diagram](https://static.igem.wiki/teams/5983/results/img2.webp)

Figure 2 Verification of HMB-Related Fragments and Single Colony Screening

(A) Agarose gel electrophoresis of PCR-amplified HMB pathway genes (B) Single colonies of engineered strains

The results show that we successfully PCR-amplified the fragments of AtoB, MvaS, AibAB, and LiuC, and cloned them into the pSB1A3 vector. Finally, single colonies were obtained on LB agar plates containing ampicillin (Amp⁺).

### HMB Production Assay

To verify the ability of the engineered strains to produce HMB, we established an HPLC detection method based on literature reports. The HPLC procedure is as follows:

First, to determine the maximum absorption wavelength (λmax) of the target compound, a full-wavelength scan was performed using an Agilent 1200 series high-performance liquid chromatography (HPLC) system equipped with a diode array detector (DAD) under the above chromatographic conditions. The detector scan range was set from 200–400 nm, with a bandwidth of 4 nm and a sampling frequency of 1 Hz, continuously collecting spectral data throughout the retention time. The UV spectrum at the peak apex of the target compound was extracted to obtain its characteristic absorption curve, allowing the determination of the maximum absorption wavelength (λmax). Based on our experiment, the detection wavelength for HMB was determined to be 208 nm.

![HMB Expression Verification Diagram](https://static.igem.wiki/teams/5983/results/img3.webp)

Figure 3 Determination of HMB Detection Wavelength

Next, HMB quantification was performed using an Agilent 1200 series HPLC system equipped with a C18 reverse-phase column (250 mm × 4.6 mm, 5 μm). The column temperature was maintained at 30°C. The mobile phase consisted of eluent A (0.02 mol/L potassium dihydrogen phosphate, pH 3.0) and eluent B (acetonitrile). The elution gradient was set as follows: initial 95% A; linear transition to 40% A from 8–8.5 min; hold at 40% A from 8.5–13.0 min; linear return to 95% A from 13.0–13.5 min; hold at 95% A from 13.5–22.0 min. The flow rate was 1.0 mL/min, the injection volume was 10 μL, and the detection wavelength was set at 208 nm (Table 1).

A series of HMB standard solutions ranging from 0.05–2.00 mg/L were measured at 208 nm to construct a standard curve, which was then used to calculate the actual HMB content in the samples (Figure 4) [1].

Table 1. HPLC Program for HMB Detection

|
|  |

Time (min) |

Eluent A |

Eluent B |

Notes |

|
|  |

0–8.0 |

95% |

5% |

Initial conditions |

|
|  |

8.0–8.5 |

95%→40% |

5%→60% |

Linear gradient |

|
|  |

8.5–13.0 |

40% |

60% |

Hold at constant composition |

|
|  |

13.0–13.5 |

40%→95% |

60%→5% |

Linear return to initial conditions |

|
|  |

13.5–22.0 |

95% |

5% |

Hold at initial conditions until the end of analysis |

![HMB Expression Verification Diagram](https://static.igem.wiki/teams/5983/results/img4.webp)

Figure 4 HMB Standard Absorption Peak at 208 nm

As an intestinal probiotic, HMB production needs to be stable and sustained. Therefore, we chose to test fermentation at 12 h as the detection time point to ensure continuous HMB production.

To evaluate the HMB yield of the engineered strains in M9 medium after 12 h, the strains were activated in LB/Amp⁺ medium and then inoculated into M9 medium. Cell growth was monitored in real time via OD₆₀₀ measurements. Samples were taken at different time points, centrifuged, and the supernatant was collected for HMB quantification. Each experiment was performed in triplicate, and Student’s t-test was used for comparison between groups, with p < 0.05 considered statistically significant.

The results showed that the engineered E. coli produced 65.13 mg/L HMB after 12 h cultivation at 37°C, whereas the wild-type BL21 did not produce detectable HMB (Figure 5).

![HMB Expression Verification Diagram](https://static.igem.wiki/teams/5983/results/img5.webp)

图5 HMB生产测试

Next, by comparing the growth curves of the wild-type and engineered strains, we evaluated the potential impact of HMB synthesis on the engineered bacteria. First, the growth of the engineered strain was not significantly inhibited, indicating that the intracellularly synthesized HMB did not exert direct growth toxicity. Second, these results suggest that the metabolic burden imposed by the introduction of the exogenous metabolic pathway was within the tolerable range of the engineered strain and did not significantly impair its normal proliferation (Figure 6).

![HMB Expression Verification Diagram](https://static.igem.wiki/teams/5983/results/img6.webp)

Figure 6 Growth Curves of HMB-Producing Strains

Finally, to quantify the carbon source utilization efficiency and the effectiveness of the metabolic pathway in the engineered strains, we dynamically monitored the glucose consumption and HMB production every 6 hours over a 24-hour period, and performed correlation analysis. The results showed a significant positive correlation between the two variables, indicating that as time progressed, HMB was continuously produced while glucose was steadily consumed (Figure 7).

This observation directly demonstrates that glucose, as the carbon source, was efficiently channeled into the de novo HMB synthesis pathway, rather than being redundantly consumed by side metabolic pathways or non-productive biomass growth, thereby validating the functional integrity of HMB production and the rationality of the engineered metabolic pathway. More importantly, these results provide core experimental data for the subsequent calculation of carbon flux efficiency and carbon source utilization rate — the efficient allocation of carbon is not only key to increasing HMB yield, but also aligns with the requirements for low-consumption, high-efficiency biosynthesis systems in resource-limited environments, such as space stations or closed ecological systems.

![HMB Expression Verification Diagram](https://static.igem.wiki/teams/5983/results/img7.webp)

Figure 7 Carbon Utilization Analysis During HMB Production

In this study, we successfully constructed engineered strains capable of HMB synthesis. The results showed that the engineered strain produced approximately 65 mg/L HMB after 12 h of cultivation in M9 medium, and HMB production did not have a significant impact on host growth. Further analysis revealed a positive correlation between HMB yield and glucose consumption, demonstrating that the engineered strain can efficiently convert carbon sources into the target metabolite.

### Thioesterase-Mediated Enhancement of HMB Production

Based on literature research and communication, thioesterases are known to enhance the conversion of HMB-CoA to HMB [2]. Therefore, we tested TesB (BBa\_25YQAEQ1), YciA (BBa\_25BAALSN), and MenI (BBa\_25A7QNVZ) from E. coli (Figure 8A, B, and C).

The results showed that upon heterologous expression of tesB, the HMB yield reached 148.32 mg/L after 12 h. Expression of YciA had the most pronounced effect, producing 185.86 mg/L HMB after 12 h. In contrast, MenI showed the lowest enhancement among the three, yielding 101.29 mg/L HMB after 12 h; however, this was still higher than the strain without thioesterase expression (Figure 8D).

![HMB Expression Verification Diagram](https://static.igem.wiki/teams/5983/results/img8.webp)

Figure 8 Thioesterase-Mediated Enhancement of HMB Production

(A) Agarose gel electrophoresis verification of tesB sequence (B) Agarose gel electrophoresis verification of YciA sequence (C) Agarose gel electrophoresis verification of MenI sequence (D) HMB production under different thioesterases

# Serotonin Expression Verification

### Strain Construction

First, we constructed a serotonin synthesis system. The serotonin-related genes TDC (BBa\_25P80OSX) and TPH (BBa\_25G48CDM) were obtained from NCBI and synthesized by Generalbial (China) after codon optimization for E. coli. Restriction sites EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI were removed to comply with RFC#10 standard and ensure compatibility with pET28a(m) cloning. The genes were then cloned into the pET28a(m) vector via the NdeI and XhoI restriction sites.

The recombinant plasmid was subsequently transformed into E. coli DH5α and BL21. Positive clones were selected on LB agar plates (1.5% agar) containing 100 μg/mL kanamycin (Kana) and sequencing verification was performed (Beijing, Qingke) to obtain the recombinant engineered strains.

![Serotonin Expression Verification Diagram](https://static.igem.wiki/teams/5983/results/img9.webp)

Figure 9 Construction of Serotonin-Producing Strains

(A) Construction of the serotonin synthesis pathway (B) Plasmid map of the serotonin synthesis construct (C) Agarose gel electrophoresis verification of serotonin-related gene amplification

### Serotonin Production Assay

To evaluate serotonin (5-HT) production, the engineered strains were inoculated at a 1:100 ratio into 20 mL of modified M9 medium, which contained 1× M9 salts (A510881, Sangon Biotech), 0.2% (w/v) glucose, 0.1% (w/v) casamino acids (C304284, Aladdin), 1 mM MgSO₄, 50 pM FeCl₃, and 50 mg/L L-tryptophan (A64769, Innochem). When the culture reached OD₆₀₀ = 0.6, 0.5 mM IPTG was added to induce gene expression, and incubation continued for 20 h.

Samples (1 mL) were collected every 5 h, filtered through a 0.22 μm hydrophilic PES membrane (BS-PES-45, Biosharp), and stored at -20°C. Serotonin concentration in the culture medium was measured using a 5-HT ELISA kit (JL53170, Jonln), and the standard curve was used to calculate the serotonin titer.

The results demonstrated that the engineered E. coli produced 1.85 mg/L serotonin, whereas the wild-type strain and the strain expressing TDC only did not produce detectable serotonin.

![Serotonin Expression Verification Diagram](https://static.igem.wiki/teams/5983/results/img10.webp)

Figure 10 Serotonin Production Assay

(A) Serotonin titer measured every 6 hours over 24 hours (B) Functional verification of serotonin-related enzymes

### Screening of TDC from Different Sources to Enhance Serotonin Production

Through interviews with experts, we learned that TDC from different sources may affect serotonin yield. Therefore, we tested TDC genes derived from rice, Catharanthus, and pig. The results showed that Catharanthus-derived TDC had the best performance, increasing the serotonin titer to 2.62 mg/L.

![Serotonin Expression Verification Diagram](https://static.igem.wiki/teams/5983/results/img11.webp)

Figure 11 Screening of TDC from Different Sources

In this study, we successfully constructed a serotonin synthesis system by cloning and expressing TDC and TPH genes in E. coli. The results showed that the recombinant strains were able to synthesize and secrete serotonin in the culture medium, with a maximum titer of 1.85 mg/L, whereas the wild-type strain and the strain expressing TDC alone did not produce detectable serotonin. Furthermore, TDC from different sources had a significant impact on serotonin yield, with Catharanthus-derived TDC performing best, increasing the titer to 2.62 mg/L.

# Construction and Verification of the Safety System

### Strain Construction

To prevent potential side effects of the engineered strains and their products in vivo, or to allow users to terminate bacterial activity on demand, we designed a safety system. An arabinose-inducible promoter (BBa\_K808000) was used to drive the expression of E. coli endogenous mRNA-cleaving toxin MazF, enabling engineered cell self-lysis in the presence of arabinose.

![Construction and Verification of the Safety System Diagram](https://static.igem.wiki/teams/5983/results/img12.webp)

Figure 12 Construction of the Safety System

(A) Genetic circuit diagram of the safety system (B) Plasmid map of the safety system construct (C) Agarose gel electrophoresis verification of safety system gene expression

### Arabinose-Inducible Promoter Assay

First, we tested the inducible function of the arabinose promoter pBAD using mRFP as a reporter. The results showed that as the arabinose concentration increased, the Fluorescence/OD₆₀₀ value continuously rose, indicating that the arabinose promoter can effectively induce protein expression in the presence of arabinose.

![Construction and Verification of the Safety System Diagram](https://static.igem.wiki/teams/5983/results/img13.webp)

Figure 13 Verification of Arabinose-Inducible Promoter Function

### Safety System Assay

Next, we synthesized the E. coli toxin gene MazF and performed codon optimization to meet the BioBrick RFC#10 standard. Using overlap PCR, the optimized mazF was placed downstream of the arabinose-inducible promoter PBAD, forming the PBAD-mazF expression cassette. This cassette was cloned into the standard plasmid vector pSB1A3 via XbaI and SpeI restriction sites and transformed into E. coli DH5α competent cells.

To evaluate the system’s effect on host growth under induction, we combined arabinose induction with growth curve monitoring. Frozen DH5α-PBAD-MazF engineered strains and wild-type DH5α controls were inoculated into 5 mL LB medium containing 100 μg/mL Ampicillin and grown overnight at 37°C, 150 rpm for activation. The next day, overnight cultures were 1:100 inoculated into 20 mL fresh LB medium with an initial OD₆₀₀ = 0.1. When cells reached OD₆₀₀ ≈ 0.4, the experimental group was induced by adding 2% (w/v) L-arabinose (A83229, Innochem) to activate PBAD-driven mazF expression.

Cell growth was continuously monitored using a FlexStation 3 microplate reader (Molecular Devices, USA) to generate growth curves. The results showed that under L-arabinose induction, the growth of DH5α-PBAD-MazF was significantly inhibited, whereas the non-induced group and wild-type controls showed no significant growth changes.

These results demonstrate that the PBAD-MazF system can effectively activate toxin expression under arabinose induction, suppressing host growth, and validate its feasibility as a controllable “suicide switch” for biosafety control in synthetic biology.

![Construction and Verification of the Safety System Diagram](https://static.igem.wiki/teams/5983/results/img14.webp)

Figure 14 Verification of the Suicide System

In summary, to prevent potential side effects of the engineered strains and their products in vivo, or to allow users to terminate bacterial activity on demand, we designed a controllable safety system. This system uses the arabinose-inducible promoter PBAD (BBa\_I13453) to drive the expression of the E. coli toxin MazF, enabling engineered cell self-lysis in the presence of arabinose.

# Summary

We successfully constructed the BL21-HMB engineered strain capable of de novo HMB synthesis. The results showed that this strain produced approximately 65 mg/L HMB after 12 h cultivation in M9 medium, with HMB production positively correlated with glucose consumption and no significant effect on host growth. Furthermore, by introducing the thioesterase YciA, HMB titer was enhanced to 185.86 mg/L, significantly improving synthetic efficiency.

Next, we established a serotonin synthesis system by successfully cloning TDC and TPH genes into E. coli. The recombinant strains were able to synthesize and secrete serotonin in the culture medium, reaching a maximum titer of 1.85 mg/L. TDC from different sources had a significant impact on serotonin yield, with Catharanthus-derived TDC increasing the titer to 2.62 mg/L.

Finally, to ensure the biosafety of the engineered strains, we designed an arabinose-inducible MazF suicide system. Under arabinose induction, this system could be activated to trigger self-lysis of engineered cells, enabling controllable regulation of growth and function.

In conclusion, we not only achieved efficient synthesis of HMB and serotonin, but also established a controllable safety mechanism, laying a solid foundation for the application of synthetic biology in health intervention and functional probiotic development.

# Reference

[1] Zhang, W.-h., Xi, C., Yi, X., Zhang, Y., Bao, W., Shao, L.-j. and Xu, D.-m. (2024), Determination of β-Hydroxy-β-Methylbutyrate Content in Food Raw Materials by High-Performance Liquid Chromatography. Sep Sci plus., 7: e202400144.

[2] Huang, S. J., Lai, M. J., Chen, A. Y., & Lan, E. I. (2024). De novo biosynthesis of 3-hydroxy-3-methylbutyrate as anti-catabolic supplement by metabolically engineered Escherichia coli. Metabolic engineering, 84, 48–58.

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