iGEM 2025 — Team subcat-united

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{% block title %}Notebook{% endblock %}
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notebook
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{% set table\_data = [{
"title": "Overview：",
"content": '

The objective of our project is to explore the
potential of enzyme preparations in antioxidant and skin anti-aging products. Therefore, through information research,
we confirmed that SOD enzyme and CAT enzyme would be our research subjects. Based on the DBTL
(Design-Build-Test-Learn) cycle concept in engineering, this project systematically conducted research on the
construction and functional verification of the antioxidant enzyme system. The specific work included:

'
},{
"title": "Design：",
"content": '

**Abstract：**

Taking the advantages of Escherichia coli as the
expression host as a priority, we screened the SodB gene encoding superoxide dismutase (SOD) and the KatG gene
encoding catalase (CAT). To improve the efficiency of heterologous expression and protein yield, codon optimization
was carried out for the Escherichia coli expression system, while considering factors such as the secondary structure
of the gene sequence and GC content to ensure the optimal transcription and translation efficiency of the genes in the
host.

During the process of protein synthesis, codons play
a crucial role in converting genetic information into the amino acid sequence of proteins. Different species often use
different codons when translating the same amino acid, and this phenomenon is known as the species-specific preference
of codons. Although the specific mechanism for the natural formation of codon preference is not yet fully understood,
it has a significant impact on the efficiency of protein expression. Especially in the expression of recombinant
proteins, to enhance the expression level, the codon usage habits of the host species are usually optimized for the
exogenous gene sequence. This strategy is particularly crucial in heterologous protein expression systems because the
target gene comes from different species and must adapt to the translation mechanism of the host to ensure efficient
expression. In addition, codon optimization can also improve the cloning efficiency of DNA, for example, by adjusting
the CG content and reducing repetitive sequences, to achieve more efficient gene construction. At the same time, codon
optimization can enhance the stability of mRNA, improve the efficiency of transcription and translation, and thereby
comprehensively promote the expression of the target protein. Based on the original gene sequence of sodB and KatG, we
optimized its sequence through an online codon optimization platform. On this basis, we carried out subsequent related
work. We chose the pET28a vector for integration construction.

![](https://static.igem.wiki/teams/6005/engineer/1.webp)

Fig .1 Design and SnapGene diagram of pET28a-sodB and
pET28a-KatG

'
}, {
"title": "Build",
"content": '

**Abstract：**

Based on molecular cloning technology, the optimized
SodB and KatG genes were respectively cloned into the high-expression vector pET28a, and recombinant plasmids were
constructed. Through transformation, the screening and amplification of Escherichia coli engineering strains, a stable
and efficient recombinant strain library was established. Subsequently, using the IPTG induction expression system,
the expression conditions were optimized to obtain highly pure and fully functional recombinant enzyme proteins,
laying the foundation for subsequent functional determination.

The sodB (SOD, GeneID: 944953) and katG (CAT, GeneID:
948431) sequences were retrieved and codon-optimized for efficient expression in Escherichia coli. The genes were
synthesized and cloned into the pET28a vector to generate recombinant plasmids, which were validated using standard
molecular biology techniques. As shown in Figure 2, target genes SOD and CAT, along with the plasmid backbone, were
successfully amplified![图片包含 黑暗, 夜晚, 站, 屏幕  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/6005/engineer/2.webp).

Fig.2 Agarose gel electrophoresis of target genes and
linearized vector: SOD, CAT and pET28a linearized plasmid.

**Transformation using Escherichia coli DH5α,
followed by monoclonal verification and DNA sequencing**

After PCR amplification, the target DNA fragments
were carefully excised from an agarose gel and purified to isolate the desired gene segments. The SOD and CAT genes
were then gently cloned into the pET28a plasmid vector using homologous recombination—a smooth and precise method that
helps ensure accuracy. The resulting recombinant plasmids were introduced into competent Escherichia coli DH5α cells,
which were plated on kanamycin-containing selective medium and incubated overnight. This step allowed us to identify
positive clones with care and confidence, bringing us one step closer to our goal.

Individual colonies from monoclonal cultures were
selected for PCR verification. Electrophoresis results (Fig. 3) showed clear, strong bands matching the expected PCR
products, confirming initial plasmid construction success. Positive clones were cultured for expansion and verified by
Sanger sequencing, which confirmed the accuracy and integrity of the recombinant plasmids.

![图形用户界面  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/6005/engineer/3.webp)

Fig. 3 Verification of pET28a-SOD and pET28a-CAT
plasmid construction by colony PCR and Sanger sequencing

Building on this foundation, the recombinant plasmid
was gently introduced into competent Escherichia coli BL21(DE3) cells. As shown in the Fig. 4, Through careful
screening on antibiotic-containing plates, we successfully identified positive clones, which were then confirmed by
colony PCR. With confidence in their accuracy, these engineered strains were carried forward for target protein
expression and functional validation, marking an important step toward our research goals.

![电脑的屏幕  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/6005/engineer/4.webp)

Fig .4 Transformation of BL21 competent cells and
verification by colony PCR

'
}, {
"title": "Test",
"content": '
',
"children":[{
"title": "Abstract",
"content": '

Strictly combining biochemical and biophysical
methods, a series of in vitro functional evaluations were carried out on the purified SOD and CAT proteins. Through
enzymatic activity detection, the scavenging efficiency of superoxide anions and hydrogen peroxide was quantified.
Further, orthogonal experiments were conducted to optimize the enzyme ratio, pH, temperature, etc., to systematically
characterize the synergistic antioxidant performance of the dual enzyme system under different environmental
conditions and establish a reliable functional evaluation system.

For the well-constructed engineered bacterial
strains, our next goal is to test and evaluate the production and expression of SOD and CAT enzymes based on the
design objectives of the project. We will further conduct tests and research on the effects of single enzyme activity
and different environmental factors on the activity of double enzymes.

'
},{
"title": "1. SDS-PAGE was used to identify the protein
expression of pET28a-SOD and pET28a-CAT.",
"content": '

Firstly, we conducted large-scale cultivation and
induction expression of the obtained engineered bacteria and extracted the cell proteins through ultrasonic
disruption. At the same time, for the crude protein obtained from the protein supernatant, we used a His-tag
purification column to purify the target protein and then analyzed the effect of protein purification through SDS-PAGE
electrophoresis. The results are shown in Figure 5. We successfully induced the expression of the constructed
BL21-pET28a-SOD and BL21-pET28a-CAT and used gradient imidazole for purifying the impurities and eluting the target
protein.

![post_object_image_1379271822](https://static.igem.wiki/teams/6005/engineer/5.webp)

Fig. 5 SDS-PAGE gel electrophoresis was used to
analyze the expression of SOD and CAT proteins, and nickel column affinity chromatography was employed.

'
},{
"title": "2. BCA assay to measure the concentration of
purified SOD and CAT",
'content': '

The concentrations of the purified SOD and CAT
proteins were determined by BCA assay using a standard curve based on absorbance values, providing key parameters for
subsequent enzyme activity assays. The two purification batches yielded CAT concentrations of 60.94 μg/mL and 144.25
μg/mL, and SOD concentrations of 83.95 μg/mL and 94.07 μg/mL.![post_object_image_1257658138](https://static.igem.wiki/teams/6005/engineer/6.webp)

Fig. 6 BCA assay for the test of protein
concentration

'
},{
"title": "3. WST-8 assay for the detection of SOD
enzyme activity",
"content": '

SOD activity was evaluated using the WST-8 assay due
to its high sensitivity and stability. As shown in Figure 7, Blank1 (positive control) contained a complete system for
generating superoxide anions, while Blank2 (negative control), lacking this system, produced no detectable signal. The
target protein group showed strong superoxide scavenging activity, with clearance efficiency close to that of the
negative control, confirming robust SOD activity. These results support further analysis of dual-enzyme synergy.

![图表  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/6005/engineer/7.webp)

Fig. 7 Assay of superoxide dismutase (SOD) enzyme
activity

'
},{
"title": "4. Hydrogen peroxide removal assay for the
detection of CAT enzyme activity",
"content": '

CAT activity was measured using a catalase activity
detection kit, which indirectly quantifies enzyme activity by detecting residual hydrogen peroxide and its chromogenic
product. As shown in Figure 8, the Blank group (negative control) lacked components to decompose hydrogen peroxide,
resulting in the highest absorbance. In contrast, both purified CAT samples reduced hydrogen peroxide levels by
approximately 50%, demonstrating significant catalytic activity. These results support subsequent dual-enzyme
co-activity assays.![图表, 折线图  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/6005/engineer/8.webp)

Fig. 8 The determination of catalase activity

'
},{
"title": "5. The superoxide anion scavenging experiment
verified the antioxidant capacity of the dual enzyme system",
"content": '

A three-factor, three-level orthogonal experiment was
used to evaluate the effects of SOD/CAT ratio, temperature, and pH on free radical scavenging efficiency. Based on
mean efficiency and range analysis (R), temperature had the greatest influence (R = 74.38), with peak performance at
25°C. Efficiency dropped sharply at 45°C, with some negative values indicating thermal inhibition. pH showed the
second-largest effect (R = 13.56), with optimal activity at pH 8.0 and lowest at pH 7.0. The SOD/CAT ratio had the
smallest impact (R = 11.03); the 1:1 ratio yielded the highest and most consistent efficiency, while 0.5:1 resulted in
high variability and frequent negative values, suggesting instability. A 2:1 ratio gave moderate results. The optimal
conditions were determined to be 1:1 SOD/CAT ratio, 25°C, and pH 8.0, under which the system achieved maximum
scavenging efficacy. These results provide a solid basis for optimizing antioxidant enzyme systems.

![表格  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/6005/engineer/9.webp)

Fig. 9 Orthogonal experiments verify the clearance of
superoxide anions

'
},{
"title": "6. ABTS+ Radical Scavenging
Activity",
"content": '

This assay used a three-factor, three-level
experiment to examine how SOD/CAT ratio, temperature, and pH affect antioxidant ability measured by Trolox equivalent
antioxidant capacity (TEAC). Results showed temperature had the biggest impact, followed by pH, while SOD/CAT ratio
had a smaller effect. Antioxidant activity was highest at 25°C and better at pH 7.0 and 8.0 than at pH 6.0, indicating
cooler and neutral-to-alkaline conditions are best. The 2:1 SOD/CAT ratio gave slightly better results than 1:1, but
both worked well. The best combination was SOD/CAT = 2:1, temperature 25°C, and pH 7.0, reaching a TEAC of 0.6519.
These results guide future experiments and process control, stressing careful temperature and pH management and
flexible SOD/CAT ratios as needed.

![图形用户界面, 应用程序  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/6005/engineer/10.webp)

Fig. 10 Orthogonal experiments verified the clearance
of ABTS+

'
}]
},{
"title": "Learn",
"content": '

Based on the experimental data analysis, the
intrinsic relationship between gene optimization, expression level, and enzyme activity was deeply analyzed. The
sensitivity of process parameters was scientifically summarized, forming a theoretical and technical framework for
enzyme preparations in antioxidant and skin anti-aging applications. The research results not only verified the
feasibility of the recombinant enzyme system but also provided data support and guidance for subsequent protein
engineering modifications, improvement of enzyme catalytic efficiency and stability, and laid a foundation for the
development and industrial application of functional enzyme preparations.

'
}]%}
{% block title %}Engineering{% endblock %}
{% block lead %}
engineering
{% endblock %}

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'
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{% block title %}Experiments{% endblock %}
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experiments
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{% extends "layout.html" %}
{% set table\_data = [{
"title": "Overview",
"content": '

With the advancement of biotechnology and increasing
demand for effective anti-aging solutions, the cosmetics and skincare industries have seen a improved development
toward products that target cellular oxidation. The free radical theory of aging identifies reactive oxygen species
(ROS) as major leading factors to cellular aging. In response, catalase (CAT) and superoxide dismutase (SOD) have been
widely studied for their role in mitigating oxidative damage. This project aims to develop a novel method to
investigate CAT and SOD by enhancing the expression and combined activity of SOD and CAT enzymes using synthetic
biology techniques. The genes encoding SOD and CAT were inserted into a plasmid vector and expressed in
*E.coli*. Following bacterial transformation, colonies were screened, and positive clones were identified
through PCR amplification and agarose gel electrophoresis. Enzymatic activity assays were conducted to confirm the
expression and functions of the enzymes. The invention of this method largely improved and emphasized the
effectiveness of the formation of SOD and CAT. The engineered SOD-CAT complex demonstrated a significance antioxidant
activity, highlighting the potential for improved efficacy in neutralizing superoxide anions.

'
},{
"title": "Plasmids construction of sodB and katG gene",
"content": '

**1. Amplification and Verification of the
targeted Gene and plasmid backbone**

First, the nucleotide sequences of sodB (SOD, GeneID:
944953) and katG (CAT, GeneID: 948431) were retrieved from the database and subjected to codon optimization
specifically designed for efficient expression in Escherichia coli. Gene synthesis was then performed based on the
optimized sequences. Subsequently, the pET28a plasmid vector was selected for cloning, and recombinant constructs were
generated using the synthesized gene fragments. The resulting plasmids were further validated through standard
molecular biology techniques. The results are presented in Figure 1: We successfully amplified the target genes SOD
and CAT, along with the plasmid backbone fragments.

![图片包含 黑暗, 夜晚, 站, 屏幕  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/6005/result/1.webp)

Fig.1 Agarose gel electrophoresis of target genes
and linearized vector: SOD, CAT and pET28a plasmid.

**2. Transformation using Escherichia coli
DH5α, followed by monoclonal verification and DNA sequencing**

Following PCR amplification, the target DNA fragments
were excised from an agarose gel and purified to obtain the desired gene segments. Subsequently, the SOD and CAT genes
were individually cloned into the pET28a plasmid vector via homologous recombination. The resulting recombinant
plasmids were transformed into Escherichia coli DH5α competent cells, which were then plated on selective solid medium
containing kanamycin and incubated overnight for the identification of positive clones.![图形用户界面  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/6005/result/2.webp)

Fig. 2 Verification of pET28a-SOD and pET28a-CAT
plasmid construction by colony PCR and Sanger sequencing

Individual colonies from the obtained monoclonal
cultures were selected for PCR verification. Electrophoresis results, as shown in Figure 2, revealed clear and intense
bands corresponding to the expected PCR products, indicating preliminary success in plasmid construction. Positive
clones were subsequently expanded through culturing and subjected to Sanger sequencing for further validation. The
sequencing results confirmed both the accuracy and integrity of the recombinant plasmid constructs.

![电脑的屏幕  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/6005/result/3.webp)

Fig .3 Transformation of BL21 competent cells and
verification by colony PCR

Subsequently, for the two successfully constructed
plasmids, we respectively introduced them into the competent cells of Escherichia coli BL21(DE3), and after screening
on solid plates, we confirmed the final positive engineered bacteria through colony verification.

'
}, {
"title": "Protein expression and purification",
"content": '

**1. Induction and expression of pET28a-SOD and
pET28a-CAT**

Following successful construction of the expression
vector, it was transformed into Escherichia coli BL21 (DE3) competent cells. The target engineered strains,
BL21-pET28a-SOD and BL21-pET28a-CAT, were selected through culturing on solid medium and confirmed by colony PCR.
These strains were then subjected to large-scale cultivation, followed by induction of protein expression with 1 mM
IPTG. After cell lysis via ultrasonic disruption, the recombinant proteins were purified using His-tag affinity
chromatography, yielding high-purity SOD (molecular weight approximately 21,289 Da) and CAT (molecular weight
approximately 80,054 Da).

![post_object_image_1379271822](https://static.igem.wiki/teams/6005/result/4.webp)

Fig. 4 SDS-PAGE gel electrophoresis was used to
analyze the expression of SOD and CAT proteins, and nickel column affinity chromatography was employed.

As shown in the SDS-PAGE results in Figure 4,
gradient elution with increasing imidazole concentrations (20 mM, 50 mM, 100 mM, and 150 mM) was employed during the
purification process to effectively remove contaminating proteins. The target proteins were enriched through three
consecutive elutions using 500 mM imidazole. The collected fractions from the 500 mM imidazole elution contained
highly purified target proteins, which were subsequently used as samples for enzyme activity assays.

**2. BCA assay to measure the concentration of
SOD and CAT**

![post_object_image_1257658138](https://static.igem.wiki/teams/6005/result/5.webp)

Fig. 5 Determination of protein concentration by BCA
method

The protein concentrations of the purified target
proteins were determined using the BCA assay. A standard curve was constructed to calculate the precise concentrations
of SOD and CAT based on sample absorbance values, which served as critical parameters for subsequent enzyme activity
assays. The elution fractions from two independent purification batches yielded CAT concentrations of 60.94 μg/mL and
144.25 μg/mL, respectively, while SOD concentrations were determined to be 83.95 μg/mL and 94.07 μg/mL in the
corresponding batches.

'
}, {
"title": "Functional test of the enzyme",
"content": '

**1. Detection of SOD enzyme activity by WST8
method**

![图表  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/6005/result/6.webp)

Fig. 6 Assay of superoxide dismutase (SOD) enzyme
activity

To evaluate SOD enzyme activity individually, the
WST-8 assay was utilized owing to its high sensitivity and stability. As shown in Figure 6, Blank1, serving as the
positive control, contained a complete enzymatic reaction system capable of generating abundant superoxide anions. In
contrast, Blank2, which lacked a functional superoxide anion generation system, produced no detectable signal and thus
functioned as the negative control. The target protein treatment group exhibited significant superoxide anion
scavenging activity, with clearance efficiency approaching that of the negative control, indicating robust enzymatic
activity of the purified SOD. These results establish a solid foundation for the subsequent characterization of
dual-enzyme synergistic effects.

**2. The activity of CAT enzyme was detected by
the hydrogen peroxide removal method.**

The activity of CAT enzyme was assessed using a
catalase (CAT) activity detection kit. This method indirectly measures CAT enzymatic activity by quantifying the
residual hydrogen peroxide and its corresponding chromogenic product in the reaction system. As shown in Figure 7, the
Blank group, serving as the negative control, contained no components capable of decomposing hydrogen peroxide,
resulting in the highest hydrogen peroxide concentration and consequently the highest absorbance value. In contrast,
both purified CAT protein samples demonstrated significant catalytic activity, reducing hydrogen peroxide levels by
approximately 50%. These findings provide strong support for subsequent dual-enzyme co-activity assays. ![图表, 折线图  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/6005/result/7.webp)

Fig. 7 The determination of catalase activity

As shown in Figure 7, the absorbance of enzymatic
products derived from residual hydrogen peroxide was measured at 520 nm to assess hydrogen peroxide levels, clearance
capacity, and catalase activity. Compared with the blank control group, the purified CAT protein reduced hydrogen
peroxide concentration by approximately 50%. Furthermore, both replicate preparations of purified CAT exhibited
consistent enzymatic activity, indicating high batch-to-batch stability of the purified enzyme. This assay confirmed
the functional activity of CAT as a single enzyme and provides a basis for its subsequent application in combination
with other enzymes.

**3. Orthogonal Experiment 1: Superoxide Anion
Scavenging (Effects of Temperature, pH, and SOD/CAT Ratio)**

![表格  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/6005/result/8.webp)

Fig. 8 Orthogonal experiments verify the clearance of
superoxide anions

A three-factor, three-level orthogonal experiment was
conducted to systematically evaluate the effects of SOD/CAT ratio, temperature, and pH on free radical scavenging
efficiency. By calculating the mean scavenging efficiency at each factor level and analyzing the corresponding ranges
(R), it was determined that temperature exerted the most significant influence (R = 74.38). Specifically, the
scavenging efficiency at 25°C was markedly higher than at 37°C and 45°C. At elevated temperature (45°C), a substantial
decline in efficiency was observed, with some measurements yielding negative values, indicating thermal inhibition of
antioxidant activity. The pH value exhibited the second-largest effect (R = 13.56), with alkaline conditions (pH 8.0)
favoring enhanced scavenging performance, whereas pH 7.0 resulted in the lowest efficiency among the tested levels.
The SOD/CAT ratio had a comparatively minor impact (R = 11.03). A ratio of 1:1 demonstrated the highest scavenging
efficiency and the greatest consistency across replicates, while a ratio of 0.5:1 showed pronounced variability and
frequent negative values, suggesting poor stability. A ratio of 2:1 yielded moderate but less optimal performance.
Overall, the optimal condition was identified as an SOD/CAT ratio of 1:1, a temperature of 25°C, and a pH of 8.0,
under which the system achieved peak scavenging efficacy. These findings provide a robust experimental basis for
parameter optimization in the design of efficient antioxidant enzyme systems.

**4. Orthogonal Experiment 2: ABTS+
Radical Scavenging Activity (Effects of Temperature, pH, and SOD/CAT Ratio)**

![图形用户界面, 应用程序  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/6005/result/9.webp)

Fig. 9 Orthogonal experiments verified the clearance
of ABTS+

Based on a three-factor, three-level orthogonal
experimental design, a systematic evaluation was conducted to investigate the effects of SOD/CAT ratio, temperature,
and pH on antioxidant capacity, as quantified by the Trolox equivalent antioxidant capacity (TEAC) assay. By
calculating the mean TEAC values across different levels of each factor and comparing their respective ranges (R), the
results revealed that temperature exerted the most significant influence on antioxidant activity, followed by pH,
whereas the SOD/CAT ratio had a relatively minor yet discernible effect. Specifically, the experimental data
demonstrated that antioxidant capacity at 25°C was significantly higher than at 37°C and 45°C. Moreover, performance
at pH 7.0 and 8.0 was superior to that at pH 6.0, indicating that lower temperatures and neutral-to-alkaline
conditions are more favorable for enhancing antioxidant efficacy. With respect to the SOD/CAT ratio, although the
highest average TEAC value was observed at a ratio of 2:1, the difference compared to the 1:1 ratio was modest (range
≈ 0.1), suggesting that both ratios contribute to antioxidant activity with only limited variation between them.
Overall, the optimal combination of conditions was determined to be an SOD/CAT ratio of 2:1, a temperature of 25°C,
and a pH of 7.0, under which the TEAC value reached its maximum of 0.6519. These findings provide a scientific basis
for subsequent experimental design and process optimization. Furthermore, the results highlight the critical
importance of precise control over temperature and pH, while allowing for flexible selection of the SOD/CAT ratio
depending on practical requirements.

'
},{
"title": "Summary",
"content": '

Based on the present study, superoxide dismutase
(SOD) and catalase (CAT) have been successfully expressed, purified, and functionally validated as active enzymes in
vitro. The antioxidant performance of the dual-enzyme system under varying conditions has been systematically assessed
through orthogonal experimental design. Future research may be directed toward the following aspects: First, given
that superoxide anion scavenging activity is optimal at 25°C, pH 8.0, and an SOD/CAT ratio of 1:1, further
investigation into the system’s stability and kinetic behavior across broader temperature and pH ranges is recommended
to enhance its applicability under diverse environmental or physiological conditions. Second, since the highest ABTS⁺
radical scavenging efficiency was observed at an SOD/CAT ratio of 2:1, 25°C, and pH 7.0, it is essential to elucidate
the underlying synergistic mechanisms between the two enzymes, particularly the minimal performance variation between
the 1:1 and 2:1 ratios, which could inform rational enzyme proportion optimization and cost-effective formulation
development. Third, in vivo relevance should be evaluated by extending in vitro findings to cellular and animal models
to assess the therapeutic efficacy and biosafety of the dual-enzyme system, thereby validating its translational
potential. Finally, strategies such as protein engineering or advanced delivery systems (e.g., nanocarriers or
targeted vectors) should be explored to improve enzymatic stability, bioavailability, and site-specific targeting,
better meeting clinical or industrial requirements for oxidative stress intervention. Collectively, these research
directions will provide a solid theoretical foundation and technical support for the rational design and application
of dual-enzyme antioxidant systems.

'
}]%}
{% block title %}Results{% endblock %}
{% block lead %}
results
{% endblock %}