iGEM 2025 — Team mca

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|  |  |
| --- | --- |
| Date | Contents |
| Aug 6-7 | 1. Prepared liquid and solid LB medium 2. Prepared antibiotic stock solutions 3. Defined PCR system and conditions 4. Made 1% agarose gel 5. Performed inoculation for bacterial liquid culture |
| Aug 8-12 | 1. Extracted and purified pET28a and pETDuet-1 plasmids 2. Carried out double digestion with a restriction enzyme 3. Performed agarose gel electrophoresis and gel extraction 4. Performed homologous recombination and transformation (partial failure, requiring re-preparation) |
| Aug 13-16 | 1. Observed colony growth for qualitative detection 2. Conducted colony PCR to verify recombinant plasmids 3. Extracted pETDuet-AGE plasmids 4. Conducted enzyme digestion, gel extraction, homologous recombination, and transformation 5. Prepared reagents and systems for PCR |
| Aug 17-20 | 1. Observed colony morphology and growth status 2. Conducted colony PCR and gel electrophoresis 3. Checked inoculated colony status (partial failure, requiring repetition) 4. Cultured colonies for protein analysis 5. Prepared SDS-PAGE gel |
| Aug 21-23 | 1. Extracted plasmids and sent them for sequencing (re-cultured some samples) 2. Obtained BL21 bacterial pellets 3. Performed ultrasonic cell disruption and collected the supernatant 4. Conducted protein purification 5. Prepared samples and reagents for SDS-PAGE and enzyme activity assay |
| Aug 24-25 | 1. Completed SDS-PAGE, followed by staining and destaining 2. Determined AGE enzyme activity and performed thin-layer chromatography (TLC) analysis |
| Aug 26-27 | 1. Performed TLC analysis for NAL-catalyzed reactions (failed due to deteriorated running buffer) 2. Prepared materials and conditions for whole-cell biocatalysis 3. Conducted ELISA experiment (control group requiring repetition) |
| Aug 28-29 | 1. Expanded bacterial culture and induced protein expression 2. Collected and washed cells 3. Optimized the formulation of the running buffer 4. Analyzed TLC results of samples with different reaction times |
| Aug 30 | 1. Clarified task assignments for each team member 2. Designed the academic defense PPT and practiced once |
| Aug 31 | 1. Conducted a simulated academic defense to collect feedback for further improvements |
| Middle September | 1. Each team member proceeded with their designated tasks and drafted the wiki content under their    responsibility |
| End of September-Middle October | 1. Conduct two rounds of mock defenses to adapt to the process and ensure full preparation for the formal    academic defense |

',
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{% block title %}Notebook{% endblock %}
{% block lead %}NOTEBOOK{% endblock %}

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{
"title": 'Overview',
"content": '

NeuAc has a variety of functions, including promoting infants’ neurodevelopment and
anti-inflammatory ability. We use two enzymes, AGE and NAL, to catalyze GlcNAc to form NeuAc. We designed plasmids
that can express the enzymes, including optimization of codons and addition of several parts necessary for the
expression of our target gene. These two enzymes will then be expressed by the plasmids, induced by IPTG, and
their activity levels will finally be examined through functional tests. The entire process will be completed
inside the bacteria using the method of whole-cell biocatalysis. The specific process is shown below (Figure 1),
covering the engineering of target plasmids, protein expression, and functional tests.

**![c4832c94e25268c101d26cd084581cb](https://static.igem.wiki/teams/5766/engineering/1.webp)**

**Figure 1 The detailed process of the whole experiment**

',
"children": []
},
{
"title": 'Cycle 1: pETDuet-AGE-NAL',
"content": '

**Design:**

To construct the recombinant plasmid pETDuet-AGE-NAL, the plasmid pETDuet-AGE was first constructed to serve as
the vector. The plasmid pETDuet-AGE was constructed by ligating the AGE with pETDuet, as shown in Figure 2. The
plasmid carries a ribosome-binding site, His-tag, S-tag, AmpR gene, lac operon, as well as T7 promoter and
terminator. The AmpR gene is used for selection via antibiotics. Homologous recombination was employed to
construct pETDuet-AGE. This plasmid functions as an IPTG-inducible expression vector and contains two core
regulatory elements: the lac operon and T7 promoter. The lac operon binds the lac repressor to block
transcription, whereas the T7 promoter drives high-level protein expression upon IPTG induction and provision of
T7 RNA polymerase. These elements jointly regulate the expression of downstream proteins tagged with His and S,
which are used for subsequent protein purification.

![](https://static.igem.wiki/teams/5766/engineering/2.webp)

**Figure 2 The plasmid map of pETDuet-AGE**

After obtaining pETDuet-AGE, pETDuet-AGE-NAL was constructed by ligating the NAL fragment with pETDuet-AGE using
the same method. The plasmid map is shown in Figure 3 below.

![](https://static.igem.wiki/teams/5766/engineering/3.webp)

**Figure 3 The plasmid map of pETDuet-AGE-NAL**

**Build:**

After designing the plasmid types used for expressing the protein, we first amplified the DNA inside using PCR
technology. The sequences of AGE (1262 bp) and NAL (987 bp) were amplified with high specificity, showing single,
discrete bands upon electrophoresis, as shown in Figure 4.

**![3f4f64d3ee7dbc71e5a31defeaba5b6f.jpg](https://static.igem.wiki/teams/5766/engineering/4.webp)**

**Figure 4 Gel electrophoresis of PCR amplified AGE and NAL.**

To construct the recombinant plasmid pETDuet-AGE, we first performed restriction enzyme digestion to linearize the
pETDuet-1 vector. In the electrophoresis results (Figure 5), all pETDuet-1 samples digested with NcoI/BamHI showed
at least one clear and intact band. This indicates the successful linearization of the vector.

**![双酶切组图_画板 1](https://static.igem.wiki/teams/5766/engineering/5.webp)**

**Figure 5 Double enzyme digestion of plasmid pETDuet**

Prior to constructing the pETDuet-AGE-NAL plasmid, we first completed the construction of pETDuet-AGE. This
construction employed a homologous recombination approach. The colonies obtained after transformation are shown in
Figure 4, indicating successful recombination and the acquisition of positive clones. Subsequently, we selected a
single colony for colony PCR verification. Electrophoresis revealed a specific band at 1262 bp, matching the
expected size, preliminarily confirming that the AGE fragment had been correctly recombined into the pETDuet
vector. To further validate the construction accuracy, we extracted the recombinant plasmid and submitted it for
sequencing. Sequence alignment confirmed that the AGE gene was correctly inserted with a fully intact reading
frame, showing no frameshifts or mutations. This indicates the successful construction of the pETDuet-AGE plasmid,
laying the foundation for subsequent introduction of the NAL gene and further dual-enzyme co-expression studies.

**![AGE验证图_画板 1](https://static.igem.wiki/teams/5766/engineering/6.webp)**

**Figure 6 pETDuet-AGE Plasmid Verification Diagram**

The same method was used to construct the plasmid pETDuet-AGE-NAL. As shown in Figure 7, the pETDuet-AGE plasmids
digested with NdeI/XhoI showed at least one clear and intact band. This indicates the successful linearization of
pETDuet-AGE.

**![双酶切组图_画板 1](https://static.igem.wiki/teams/5766/engineering/7.webp)**

**Figure 7 Double enzyme digestion of plasmid pETDuet-AGE**

Building upon the successful construction of the pETDuet-AGE plasmid, we further employed homologous recombination
to insert the NAL gene into the vector, thereby constructing the pETDuet-AGE-NAL recombinant plasmid. Clear single
colonies were visible on the transformation plates, indicating successful recombination. Randomly selected
colonies were subjected to PCR validation. Electrophoresis analysis of the amplified products revealed a specific
band at the expected size (approximately 2249 bp), preliminarily confirming the correct insertion of the NAL gene.
Subsequently, positive clone plasmids were extracted for sequencing analysis. The sequencing results fully matched
the expected sequence, confirming that both the AGE and NAL genes were correctly integrated into the pETDuet
vector with intact reading frames and no frameshift mutations. These results indicate the successful construction
of the pETDuet-AGE-NAL plasmid, providing a reliable vector for subsequent dual-enzyme co-expression and catalytic
function studies.

![AGE+NAL组图_画板 1_画板 1](https://static.igem.wiki/teams/5766/engineering/8.webp)

**Figure 8 pETDuet-AGE-NAL Plasmid Verification Diagram**

**Test:**

Protein expression was induced with IPTG (1 mM) in *E. coli* BL21(DE3), followed by cell lysis using a
sonicator under cold conditions to preserve enzyme activity. The recombinant protein was purified using nickel
affinity chromatography via its 6×His-tag. The purification process involved washing with low (buffer A) and high
(buffer B) concentrations of imidazole. The collected fractions were analyzed by SDS-PAGE. As shown in Figure 9, a
distinct protein band was observed in the elution solution, indicating that our plasmid successfully expresses the
target protein.

**![](https://static.igem.wiki/teams/5766/engineering/9.webp)**

**Figure 9 SDS-PAGE of pETDuet-AGE-NAL**

**Learn:**

The plasmid pETDuet-AGE was constructed primarily as an intermediate vector for the subsequent assembly of
pETDuet-AGE-NAL and was not subjected to independent functional assays. Although the co-expression of non-fused
AGE and NAL enzymes from this plasmid facilitated the synthesis of sialic acid, the reaction efficiency remained
limited. ELISA quantification revealed that this configuration yielded the lowest product amount among all tested
constructs. These results suggest that while functionally active, the delocalized expression of AGE and NAL is
suboptimal. In contrast, the integrated systems—particularly those incorporating a linker or a
scaffold—demonstrate significantly higher production efficiency, making them more suitable for effective sialic
acid synthesis.

',
"children": []
},
{
"title": 'Cycle 2: pET28a-AGE-Linker-NAL',
"content": '

**Design:**

pET28a-AGE-Linker-NAL was constructed by ligating the AGE-Linker-NAL fragment with the pET28a plasmid, where
pET28a served as the vector. As a widely used prokaryotic expression vector, pET28a is primarily designed for
high-level recombinant protein expression in *E. coli*. It carries a T7 promoter—tightly regulated by T7
RNA polymerase—that enables IPTG-induced protein expression. Additionally, it contains antibiotic resistance genes
(e.g., kanamycin resistance) for selecting *E. coli* transformants harboring the plasmid. Homologous
recombination was employed for the construction of pET28a-AGE-Linker-NAL. This recombinant plasmid functions as an
IPTG-inducible expression vector and contains two fundamental regulatory elements, the lac operon and the T7
promoter, with a mechanism identical to that of pETDuet-AGE. These elements control the expression of downstream
His-tagged proteins, which are used for subsequent protein purification.

![](https://static.igem.wiki/teams/5766/engineering/10.webp)

**Figure 10 The plasmid map of pET28a-AGE-Linker-NAL**

**Build:**

After designing the plasmid constructs for protein expression, we first amplified the target DNA using PCR
technology. The AGE-Linker-NAL sequence was amplified with high specificity, showing a single, discrete band in
electrophoresis, as shown in Figure 11. We can find out that the DNA fragment was approximately 2303 bp in length,
which was consistent with the predicted size. This indicated that the target gene was successfully amplified.

**![PCR2_画板 1](https://static.igem.wiki/teams/5766/engineering/11.webp)**

**Figure 11 Gel electrophoresis of PCR amplified AGE-Linker-NAL.**

To construct the plasmid pET28a-AGE-Linker-NAL, restriction enzyme digestion was performed to linearize the pET28a
vector. In the electrophoresis analysis results (Figure 12), the pET28a plasmids digested with NcoI/XhoI showed at
least one clear and intact band. This indicates that the linearization of pET28a was successful.

![双酶切组图_画板 1](https://static.igem.wiki/teams/5766/engineering/12.webp)

**Figure 12 Double enzyme digestion of plasmid pET28a**

We further employed homologous recombination to sequentially assemble the AGE gene, Linker sequence, and NAL gene
into the pET28a vector, constructing the pET28a-AGE-Linker-NAL recombinant plasmid. Following the transformation,
uniformly distributed single colonies were observed on the agar plate, indicating successful recombinant
transformation. Several colonies were randomly selected for colony PCR verification. Electrophoresis results
showed a specific band at the expected size (approximately 2303 bp for the AGE-Linker-NAL fragment), preliminarily
confirming the correct insertion of the three-gene fusion into the vector. To further validate construction
accuracy, positive clone plasmids were extracted for sequencing analysis. Sequence results fully matched the
expected sequence, confirming that the AGE, Linker, and NAL elements were correctly integrated into the pET28a
vector as designed, with precise codon transitions and no frameshift mutations. These results confirm the
successful construction of the pET28a-AGE-Linker-NAL triple fusion expression vector, providing essential material
for subsequent studies investigating the regulatory role of the Linker sequence on the spatial conformation and
catalytic efficiency of the dual enzymes.

![A-L--N组图_画板 1](https://static.igem.wiki/teams/5766/engineering/13.webp)

**Figure 13 pET28a-AGE-Linker-NAL Plasmid Verification Diagram**

**Test:**

Protein expression was induced with IPTG (1 mM) in *E. coli* BL21(DE3), followed by cell lysis using a
sonicator under cold conditions to preserve enzyme activity. The recombinant protein was purified using nickel
affinity chromatography via its 6×His-tag. The purification process involved washing with low (buffer A) and high
(buffer B) concentrations of imidazole. The collected fractions were analyzed by SDS-PAGE. As shown in Figure 14,
a distinct protein band was observed in the elution solution, indicating that our plasmid successfully expresses
the target protein.

**![135a3308663bfb136cc7f5ee46441c3b.jpg](https://static.igem.wiki/teams/5766/engineering/14.webp)**

**Figure 14 SDS-PAGE of pET28a-AGE-Linker-NAL**

**Learn:**

By constructing a fusion protein (AGE-Linker-NAL), the two enzymes are co-expressed in a spatially coordinated
manner. This configuration enhances the channeling of the reaction intermediate, leading to a more efficient
synthesis of sialic acid than the system employing separate AGE and NAL enzymes.

',
"children": []
},
{
"title": 'Cycle 3: pET28a-Scaffold',
"content": '

**Design:**

The plasmid pET28a-Scaffold was constructed by ligating the Scaffold gene into the pET28a vector, shown in Figure
15. The plasmid contains a ribosome-binding site, His-tag, KanR, lac operon, as well as T7 promoter and
terminator. The KanR enables selection via antibiotics. The pET28a-Scaffold was constructed using homologous
recombination. As an IPTG-inducible expression plasmid, it harbors two key regulatory elements, the lac operon and
the T7 promoter, that function through the same mechanism as in pETDuet-AGE. These elements regulate the
expression of downstream His-tagged proteins, which facilitate subsequent protein purification.

![](https://static.igem.wiki/teams/5766/engineering/15.webp)

**Figure 15 The plasmid map of pET28a-Scaffold**

**Build:**

The Scaffold gene (775 bp) was PCR-amplified. We can find out that the DNA fragment of Scaffold is approximately
775 bp in length. The sequence of Scaffold was amplified with high specificity, showing a single, discrete band
upon electrophoresis, as shown in Figure 16.

**![PCR1_画板 1](https://static.igem.wiki/teams/5766/engineering/16.webp)**

**Figure 16 Agarose gel electrophoresis results of PCR for Scaffold**

To construct the plasmid pET28a-Scaffold, the pET28a vector was linearized by restriction enzyme digestion with
NcoI and HindIII. As shown in the electrophoresis analysis (Figure 17), the enzyme-digested pET28a plasmids
displayed at least one distinct and intact band, confirming successful linearization of the vector.

![双酶切组图_画板 1](https://static.igem.wiki/teams/5766/engineering/17.webp)

**Figure 17 Double enzyme digestion of plasmid pET28a**

We proceeded to employ homologous recombination to clone the artificial scaffold protein gene into the pET28a
vector, constructing the pET28a-Scaffold recombinant plasmid. Clear single colonies were observed on the plate
post-transformation, indicating successful transformation. Subsequently, multiple colonies were randomly selected
for colony PCR validation. Electrophoresis results showed a specific band at the expected size (approximately 755
bp), providing preliminary evidence that the Scaffold gene had been successfully inserted into the vector. To
further confirm construction accuracy, we extracted positive clone plasmids for sequencing analysis. The
sequencing results fully matched the expected sequence, confirming that the Scaffold gene was correctly integrated
into the pET28a vector with an intact coding frame, free of frameshifts or mutations. These results indicate the
successful construction of the pET28a-Scaffold plasmid, laying the foundation for subsequent studies investigating
the regulatory role of scaffold proteins in multi-enzyme assembly and metabolic pathway efficiency.

![支架组图_画板 1](https://static.igem.wiki/teams/5766/engineering/18.webp)

**Figure 18 pET28a-Scaffold Plasmid Verification Diagram**

**Test:**

Protein expression was induced with IPTG (1 mM) in *E. coli* BL21(DE3), followed by cell lysis using a
sonicator under cold conditions to preserve enzyme activity. The recombinant protein was purified using nickel
affinity chromatography via its 6×His-tag. The purification process involved washing with low (buffer A) and high
(buffer B) concentrations of imidazole. The collected fractions were analyzed by SDS-PAGE. As shown in Figure 19,
a distinct protein band was observed in the elution solution, indicating that our plasmid successfully expresses
the target protein.

![IMG_256](https://static.igem.wiki/teams/5766/engineering/19.webp)

**Figure 19 SDS-PAGE of pET28a-Scaffold**

**Learn:**

The scaffold protein mediates a structured interaction between AGE and NAL, forming a multi-enzyme complex. This
complex demonstrated the highest catalytic efficiency in sialic acid production among all tested constructs, as
quantified by ELISA.

',
"children": []
},
{
"title": 'Functional tests',
"content": '

**1. Thin-Layer Chromatography (TLC)**

The activities of AGE and NAL were qualitatively analyzed using TLC at two time points: 30 and 60 minutes. As
shown in Figure 20, in the AGE-catalyzed reactions, the band corresponding to the non-denatured enzyme at 30
minutes migrated similarly to the product reference, while the denatured enzyme at 60 minutes co-migrated with the
substrate. This indicates that 30 minutes was sufficient for optimal AGE activity under these conditions. For NAL,
the TLC results showed separation primarily at the substrate positions, suggesting that the current mobile phase
may not be ideal for resolving NAL-derived products due to their high polarity.

![](https://static.igem.wiki/teams/5766/engineering/20.webp)

**Figure 20 TLC for AGE and NAL under 30 and 60 minutes**

To further validate the generation of ManNAc and NeuAc, we optimized the developing solvent system for thin-layer
chromatography (TLC). After reacting at 42°C for 30 minutes, samples underwent two duplicate developments using
freshly prepared developing solvent, with TLC plates thoroughly dried using a hair dryer after each development.
Subsequently, TLC plates were immersed in a developing agent and heated at 130°C for 5 minutes for visualization.

Results showed that in the three distinct enzyme structures—AGE-NAL (A-N), AGE-Linker-NAL (A-L-N), and Scaffold +
NAL-AGE (S+A-N)—no bands corresponding to ManNAc or NeuAc were detected in the A-N group. In contrast, both A-L-N
and S+A-N groups exhibited distinct target product bands. This result indicates that introducing Linker or
Scaffold modules into the enzyme structure effectively prevents reversible reactions during metabolism, thereby
enhancing the synthetic efficiency of ManNAc and NeuAc.

![AGE+NAL组图_画板 1](https://static.igem.wiki/teams/5766/engineering/21.webp)

**Figure 21 TLC for AGE and NAL under 30 minutes**

We conducted systematic TLC analysis of the enzymatic activities of three enzyme constructs—AGE-NAL (A-N),
AGE-Linker-NAL (A-L-N), and Scaffold+NAL-AGE (S+A-N)—reacted for one hour at three temperature conditions: 25°C,
37°C, and 42°C. As shown in Figure 22A, the A-N construct yielded the highest amount of ManNAc in the 1-hour
incubation group, with no detectable NeuAc product formation at this time point. This indicates that A-N tends to
accumulate the intermediate ManNAc over shorter reaction times.

When incubation was extended to 2 hours, as shown in Figure 22B, both the A-L-N and S+A-N constructs exhibited
distinct NeuAc bands whose intensity approached that of the target product reference band. This indicates that
these constructs efficiently catalyze NeuAc synthesis upon extended reaction time. Compared to the 1-hour
incubation group, the 2-hour incubation group exhibited more pronounced product bands at 37°C, indicating that
appropriately extending the reaction time significantly enhances catalytic efficiency.

Collectively, these results indicate that the A-N construct favors ManNAc accumulation in short-duration
reactions, while the A-L-N and S+A-N constructs promote final NeuAc production after extended reaction times, with
37°C and 2 hours representing optimal conditions. This phenomenon indicates significant differences in catalytic
properties among different enzyme constructs. The introduction of linkers or scaffolds may promote the conversion
of reaction intermediates into end products, thereby enhancing overall catalytic efficiency.

**![全细胞催化组图_画板 1](https://static.igem.wiki/teams/5766/engineering/22.webp)**

**Figure 22 TLC for NAL-AGE (A-N), AGE-Linker-NAL (A-L-N), and Scaffold + NAL-AGE (S+A-N) under 25°C, 37°C, and
42°C for 1 hour,and 2 h**

**3.2 Enzyme-linked immunosorbent assay (ELISA)**

To evaluate the catalytic performance of the dual-enzyme system, an ELISA-based quantification assay was
conducted.

**Table 1 The standard table of Neu5Ac**

|  |  |
| --- | --- |
| Concentration (ng/mL) | Absorbance |
| 0 | 0.0889 |
| 10 | 0.5624 |
| 20 | 1.0481 |
| 40 | 1.5943 |
| 80 | 2.0665 |
| 160 | 2.3355 |

A 4-parameter logistic regression (4PL) model was employed for standard curve fitting, with the equation shown in
Figure 23, which exhibited a high correlation coefficient (R² = 0.9999).

![](https://static.igem.wiki/teams/5766/engineering/23.webp)

**Figure 23 The equation and the standard curve for the ELISA experiment**

The absorbance measured in the experiment was substituted into the equation for calculation to obtain the initial
detected concentration of sialic acid. The actual content of sialic acid in the samples was then derived by
multiplying by the dilution factor (5 times), as shown in Table 2.

**Table 2**

|  |  |  |  |
| --- | --- | --- | --- |
| Samples | Absorbance | Initial detection concentration | Actual concentration |
| Control Group | 0.0457 | 0.0000 | 0.0000 |
| A-N 1 mg/mL | 1.6396 | 41.9429 | 209.7146 |
| A-N 5 mg/mL | 0.2220 | 3.4540 | 17.2700 |
| A-N 10 mg/mL | 0.4331 | 7.4243 | 37.1216 |
| A-L-N 1 mg/mL | 0.6802 | 12.1123 | 60.5615 |
| A-L-N 5 mg/mL | 0.7649 | 13.8322 | 69.1611 |
| A-L-N 10 mg/mL | 1.0244 | 19.7337 | 98.6683 |
| S+A-N 1 mg/mL | 1.2331 | 25.5186 | 127.5932 |
| S+A-N 5 mg/mL | 0.8622 | 15.9175 | 79.5877 |
| S+A-N 10 mg/mL | 1.4269 | 32.2130 | 161.0652 |

The final results were plotted in Figure 24. For the enzyme construct A-N, the sialic acid synthesis efficiency
was the highest at a concentration of 1 mg/mL, and decreased at medium to high concentrations (5-10 mg/mL). At a
low concentration, the rate of the first-step reaction in sialic acid synthesis was slightly higher than that of
the second-step reaction, resulting in the overall reaction proceeding toward sialic acid synthesis. Under high
concentration conditions, the product sialic acid was decomposed into intermediate products by the NAL enzyme,
which were further decomposed by the AGE enzyme.

For the enzyme construct A-L-N, the content of sialic acid showed an upward trend as the concentration increased.
Compared with the maximum value of 1 mg/mL in the A-N group, the detection values at various concentrations in the
A-L-N group were relatively stable. This indicated that the role of the Linker is to stabilize enzyme function
rather than overactivate it.

For the enzyme construct S+A-N, all detection indicators were generally higher than those in the A-L-N group. At
the concentration of 10 mg/mL, the detected value was second only to that of the A-N group at 1 mg/mL. Through the
clustering effect, the Scaffold formed high-concentration A-N enzyme clusters locally, significantly improving the
reaction efficiency. Meanwhile, by binding to the AGE and NAL enzymes, the Scaffold induced their conversion from
an inactive conformation to an active conformation, which could directly enhance the catalytic efficiency of
individual enzymes. This was the core reason why the detected values of the S+A-N group were higher than those of
the A-L-N group.

**In conclusion, the enzyme construction Scaffold+AGE-NAL can maximize the efficiency of sialic acid synthesis at
medium to high concentrations, making it the optimal solution for efficiently increasing sialic acid
production.**

![](https://static.igem.wiki/teams/5766/engineering/24.webp)

**Figure 24 Concentration of Neu5Ac tested by the ELISA Kit calculated by the standard curve**

**3.3 High-Performance Liquid Chromatography (HPLC)**

The catalytic experiment of the A-N enzyme construct was illustrated in Figure 25A. The sialic acid content
reached its highest level at a reaction time of 60 minutes across all three reaction temperatures. As the reaction
time prolonged, the sialic acid yield exhibited a decreasing trend. We hypothesize that this phenomenon was
associated with the reversibility of the catalytic reaction. When the content of the intermediate product
ManNAc-6P accumulated to a certain extent, the reaction proceeded in the reverse direction. Consequently, the
substrate for the second step of the reaction was reduced, leading to a decrease in the final yield of sialic
acid. Under the condition of a reaction time of 60 minutes, the reaction temperature of 42 °C yielded the highest
accumulation of sialic acid. Overall, the optimal reaction system for enzyme construct A-N is as follows:
temperature 42 °C + reaction time 60 minutes.

The similar results were also observed when enzyme constructs A-L-N and S+A-N were used, as shown in Figure 25B
and 25C. Notably, when the S+A-N enzyme construct was used, the accumulation of sialic acid rebounded under the
conditions of 42 °C and a reaction time of 240 minutes. This may be attributed to the fact that the protein
scaffold effectively reduced the occurrence of reversible reactions under such reaction conditions. Overall, the
optimal reaction systems for both enzyme constructs A-L-N and S+A-N were as follows: temperature 42 °C + reaction
time 60 minutes.

![](https://static.igem.wiki/teams/5766/engineering/25.webp)

**Figure 25 Sialic acid production by each enzyme construct under different reaction times and
temperatures**

The sialic acid production of the three enzyme constructs under the condition of 42 °C, 240 minutes was
illustrated in Figure 26. Although the sialic acid content decreased as the reaction time prolonged, the enzyme
construct S+A-N exhibited the smallest degree of decrease and achieved the highest final accumulated product
concentration.
**This indicated that construct S+A-N can effectively reduce the occurrence of reversible reactions and was thus
the optimal enzyme construct for producing sialic acid.**

![](https://static.igem.wiki/teams/5766/engineering/26.webp)

**Figure 26 Sialic acid production by three enzyme constructs under 42 ℃, 240 minutes**

Through detailed analysis of the HPLC peak chart in Figure 27, the successful generation of sialic acid and the
significant catalytic advantage of the S+A-N construct can be further confirmed.

In the chromatogram of the S+A-N construct, a distinct and sharp peak appears at approximately 11 minutes
retention time, which aligns perfectly with the characteristic peak of sialic acid in the standard sample. This
peak exhibits symmetry and a large area, indicating high product concentration and excellent separation with no
significant tailing.

In contrast, although product peaks are detectable at the same retention time for other constructs (e.g., A-N and
A-L-N), their response intensity is significantly lower than that of S+A-N. These peaks are smaller and
accompanied by minor impurity peaks. Particularly in the A-N sample, a chromatographic peak belonging to the
intermediate ManNAc (retention time approximately 3 minutes) appeared before the target product peak, further
indicating incomplete reaction and lower catalytic efficiency.

In summary, the HPLC quantitative results align with previous activity assessment conclusions: the S+A-N construct
most effectively catalyzes sialic acid synthesis, yielding the highest product yield and optimal purity, fully
demonstrating its superiority as a highly efficient catalytic module.

![](https://static.igem.wiki/teams/5766/engineering/27.webp)

**Figure 27 Chromatograms of sialic acid content under 42 ℃, 240 minutes**

',
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{% block title %}Engineering Success{% endblock %}
{% block lead %}ENGINEERING{% endblock %}

{% extends "layout.html" %}
{% set info\_data = [
{
"title": '1 Preparation of Stock Solutions',
"content": '

**1.1 Preparation of LB Medium**

**Purpose:** To create a functional and nutritional liquid environment for bacteria to grow. Liquid
media are used for various purposes, such as the propagation of microorganisms or other tests.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Sterilized Duran bottles, with caps |
|  | Autoclave |
|  | Electronic Balance |
|  | Weighing Paper |
|  | Lab Refrigerator |
|  | Parafilm |
| Chemicals and Reagents | Tryptone |
|  | Yeast Extract |
|  | Sodium Chloride |
|  | Agar |
|  | Double-distilled H2O |

**1L of LB medium was prepared in-house with the following components:**

|  |  |
| --- | --- |
| Component | Mass |
| Tryptone | 10 g |
| Yeast Extract | 5 g |
| Sodium Chloride | 10 g |
| Double-distilled H2O | 1 L |
| Agar (to prepare a solid medium) | Add 1.5 g per 100 ml of ddH₂O |

**Procedure:**

For the Liquid media:

1) Dissolve all components in distilled H₂O.

2) Autoclave at 121°C for 20 minutes.

3) Store at 4°C (pre-use equilibration to room temperature).

For the solid media:

1) Dissolve all components in distilled H₂O.

2) Autoclave at 121°C for 20 minutes.

3) Cool to 50°C, add antibiotic.

4) Pour 15–20 mL per plate in sterile petri dishes.

5) Coagulate at room temperature, wrap in parafilm, and store inverted at 4°C.

**1.2 Preparation of Antibiotics**

**Purpose:** To establish a selective growth environment to allow only target cells to survive and
proliferate, while eliminating non-target cells that lack specific antibiotic resistance genes, thereby enabling
the accurate selection of positive clones required for the experiment.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Sterilized Duran bottles, with caps |
|  | Sterile Centrifuge Tubes |
|  | 0.22μm Filter |
|  | Disposable Syringe |
|  | Electronic Balance |
|  | Weighing Paper |
|  | Lab Refrigerator |
| Chemicals and Reagents | Antibiotic Powder |
|  | Double-distilled H2O |

**Recipes:**

|  |  |  |  |
| --- | --- | --- | --- |
| Antibiotic | Concentration | Working Concentration | Preparation |
| Kanamycin | 100mg/mL | 50 µg/mL | Dissolve 100mg in 1mL sterile H₂O |
| Ampicillin | 50mg/mL | 100 µg/mL | Dissolve 50mg in 1mL sterile H₂O |

**Procedure:**

1) Dissolve the component in distilled H₂O.

2) Filter sterilize using a 0.22 µm PVDF syringe filter.

3) Aliquot and store at -20°C.

',
"children": []
},
{
"title": '2 The construction of plasmid',
"content": '

**2.1 PCR amplification**

**Purpose:** To amplify target DNA for gel extraction/cloning.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Centrifuge tube |
|  | Centrifuge |
|  | PCR thermal cycler |
|  | Lab refrigerator |
| Chemicals and Reagents | 2×PrimeMixStar |
|  | Primer |
|  | Template DNA |
|  | ddH2O |

Reaction Mix (50 µL Total):

|  |  |
| --- | --- |
| Component | Volume (μL) |
| 2×PrimeMixStar | 25 |
| Forward Primer | 1 |
| Reverse Primer | 1 |
| Template DNA | 1 |
| ddH2O | 22 |
| Total | 50 |

Cycling Conditions (30 cycles):

|  |  |  |  |
| --- | --- | --- | --- |
| Step | Temperature (°C) | Time | Number of cycles |
| Initial Denaturation | 95 | 3 minutes | 1 |
| Denaturation | 95 | 30 seconds | 30 |
| Annealing | 56-60 | 30 seconds |
| Extension | 72 | 1 minute |
| Final Extension | 72 | 5 minutes | 1 |
| Hold | 4 | ∞ |  |

Note: Annealing temperature is primer-specific.

**Procedure:**

Mix all components in labeled PCR tubes, followed by brief centrifugation. Load samples into a pre-programmed
thermal cycler running through the PCR amplification reaction procedure above.

**2.2 Agarose gel Electrophoresis**

**Purpose:** To verify the DNA bands’ lengths and thus prepare for the next step of gel extraction to
extract the target bands.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Casting tray |
|  | Electrophoresis chamber |
|  | Microwave |
|  | UV Transilluminator / Gel Imager |
| Chemicals and Reagents | Agarose |
|  | 1× TAE Buffer |
|  | ddH2O |
|  | Nucleic acid gel stain |
|  | DNA ladder |
|  | DNA sample |
|  | Loading Buffer |

**Procedure:**

1) Dissolve 1 g agarose in 100 mL 1× TAE buffer. Heat the material in a microwave at short intervals until the
agarose dissolves completely.

2) Add 7 µL nucleic acid gel stain.

3) Pour into mold, insert comb. Solidify for 30 minutes at room temperature.

4) Mix 5 μl of PCR product with 1μl of loading buffer, followed by loading into the wells alongside the DNA
marker.

5) Perform electrophoresis at a constant voltage of 100V for 45 minutes in 1×TAE buffer.

6) After electrophoresis, place the gel in a UV gel imaging system for observation.

**2.3 Bacterial Culture and Transformation**

**Purpose:** To obtain a sufficient quantity of engineered strains with stable activity for
subsequent experiments.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Laminar flow hood |
|  | Shaking incubator |
|  | Shaking the culture tube |
| Chemicals and Reagents | LB liquid medium |
|  | antibiotic(kan+) |
|  | glycerol stock(pRSFDuet） |

**Procedure:**

1) Add 5 µL antibiotic stock (e.g., Kanamycin for pET28a) to 5 mL sterile LB.

2) Inoculate with 5 µL glycerol stock (e.g., *E. coli* BL21 with plasmid).

3) Incubate at 37°C, 220 rpm for 16–18 hours.

**2.5 Extraction of Plasmids**

**Experimental Workflow Overview:**

![9b0438a16aa4247c9719b1d533100d22.png](https://static.igem.wiki/teams/5766/experiment/1.webp)

**Goal:** To obtain high-purity plasmid DNA from host cells, including empty vectors or recombinant
vectors.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Centrifuge |
|  | Centrifugate tube |
|  | FastPure DNA Mini Column |
|  | Collection Tubes |
| Chemicals and Reagents | Buffer P1 |
|  | Buffer P2 |
|  | Buffer P3 |
|  | Buffer PW1 |
|  | Buffer PW2 |
|  | ddH2O |

**Procedure:**

1. Transfer 2 mL of the overnight culture into a 1.5 mL microcentrifuge tube. Centrifuge at 12000 rpm for 1 min,
   and drop the solution.
2. Add 250 μL Buffer P1 containing RNase A, and mix thoroughly by vortexing.
3. Add 250 μL Buffer P2, and gently invert the tube up and down 10 times to fully lyse the bacterial cells.
4. Add 350 μL Buffer P3, immediately invert gently up and down 10 times to mix thoroughly, then centrifuge at
   12,000 rpm for 2 minutes.
5. Place the FastPure DNA Mini Columns spin column into the Collection Tubes, carefully pipette the supernatant
   into the adsorption columns, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid, and put the
   adsorption column back into the collection tube.
6. Add 500 μL of Buffer PW1, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.
7. Add 600 μL of Buffer PW2, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.
8. Repeat step 7.
9. Put the spin column back into the collection tube, and centrifuge at 12,000 rpm for 1 minute.
10. Place the spin column into a new clean 1.5 mL centrifuge tube. Add 40 μL of ddH2O to the middle part
    of the adsorption membrane, let it stand at room temperature for 1 minute, then centrifuge at 12,000 rpm for 1
    minute. Measure the concentration via Nanodrop and store the plasmid DNA solution at 4 ℃.

**2.6 Restriction Enzyme Double Digestion**

**Purpose:** To double-digest the plasmid vector to linearize it, providing a backbone for
recombination and preventing self-ligation.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Centrifuge tube |
|  | Centrifuge |
|  | PCR thermal cycler |
|  | Lab refrigerator |
|  | Water bath pot |
| Chemicals and Reagents | plasmid |
|  | restriction enzyme |
|  | 10×cutsmart Buffer |
|  | ddH2O |

Reaction Mix (50 µL):

|  |  |
| --- | --- |
| Component | Volume |
| plasmid | 500 ng |
| Restriction Enzyme A | 1 µL |
| Restriction Enzyme B | 1 µL |
| 10× Buffer | 5 µL |
| ddH2O | To 50 µL |

**Procedure:**

1. Add all the components to PCR tubes, followed by brief centrifugation at 12,000 rpm for 10 seconds.

Enzyme Pairs:

pETDuet-1 + AGE insert: NcoI/BamHI

pET28a + S insert: NcoI/HindIII

2. Incubate the reaction system in a 37 ℃ water bath for 30 minutes to ensure complete digestion of the vector
   plasmid into linearized fragments. Store at 4 ℃.

**2.7 Gel Extraction and Purification**

**Purpose:** To isolate and purify linearized vector DNA fragments from agarose gel, while removing
impurities such as uncut circular vectors, other restriction-generated fragments, proteins, and agarose.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Centrifuge tube |
|  | Centrifuge |
|  | Scalpel |
|  | Lab Refrigerator |
|  | Water bath pot |
| Chemicals and Reagents | Buffer GDP |
|  | Buffer GW |
|  | ddH2O |

**Procedure:**

1. After completing agarose gel electrophoresis, precisely excise the gel slice containing linearized vector DNA
   under UV illumination. Transfer the excised gel slice to a 1.5 mL centrifuge tube.
2. Add an equal volume of Buffer GDP. Incubate at 65°C in a metal bath until the gel becomes completely
   transparent.
3. Transfer the solution to the spin column and centrifuge at 12000 rpm for 60 seconds.
4. Discard the filtrate. Place the spin column in the collection tube. Add 300 μL Buffer GDP to the spin column,
   and let it stand for 1 minute. Centrifuge at 12000 rpm for 60 seconds.
5. Discard the filtrate. Place the spin column in the collection tube. Add 700 μL Buffer GW to the spin column.
   Centrifuge at 12000 rpm for 60 seconds.
6. Repeat Step 5.
7. Discard the filtrate. Place the spin column in the collection tube. Centrifuge at 12000 rpm for 2 minutes.
8. Place the spin column into a new clean 1.5 mL centrifuge tube. Add 20 μL of ddH2O to the middle part
   of the adsorption membrane, let it stand for 2 minutes. Centrifuge at 12,000 rpm for 1 minute. Store the DNA
   solution at -20℃.

**2.8 Homologous Recombination**

**Purpose：** To construct a recombinant vector by ligating the target gene to the linearized vector
via homologous recombination.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | PCR thermal cycler |
|  | Centrifuge |
|  | Incubator |
|  | Water bath pot |
| Chemicals and Reagents | 5×CE enzyme |
|  | target gene |
|  | Linearized vector |
|  | ddH2O |

Reaction Mix (10 µL):

|  |  |  |
| --- | --- | --- |
| Component | Volume | Example |
| Linearized vector | 0.02 x bp (ng) | 6.3 kb → 126 ng |
| target gene | 0.04x bp (ng) | 500bp → 20 ng |
| 5×CE enzyme | 2 μL |  |
| ddH2O | To 10 µL |  |

**Procedure:**

1. Prepare the system:
2. Place the system in a 37°C water bath for 30 minutes, then cool to 4℃ immediately

**2.9 Qualitative Transformation Check**

**Purpose:** To quickly assess whether the transformation of target plasmids (e.g., AGE-pETDuet,
S-pET28a) into bacterial strains (*E. coli* DH5α or BL21) was successful by observing colony growth on
selective plates.

**Procedure:**

1) Following transformation, plate the cell suspension onto LB agar plates containing the appropriate
antibiotic（Kanamycin for pET28a derivatives, Ampicillin for pETDuet-1 derivatives).

2) Incubate plates overnight at 37°C.

3) Observe and document colony formation. The presence of distinct colonies indicates a successful transformation,
while no growth suggests failure.

Note: Always include a negative control (untransformed cells) to confirm the antibiotic\'s effectiveness.

',
"children": []
},
{
"title": '3.Protein expression',
"content": '

**3.1 Preparation for the gel of SDS-PAGE**

**Purpose:** To separate proteins for subsequent qualitative, quantitative, or identification
analysis of the proteins.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Electronic Balance |
|  | pH Meter |
|  | Fume Hood |
|  | Reagent Bottles |
|  | Gel Casting Plates |
|  | Gel Combs |
|  | Sealing Gaskets |
|  | Pipette |
| Chemicals and Reagents | Acrylamide |
|  | N, N\'-Methylenebisacrylamide |
|  | 1.5M Tris-HCl Buffer |
|  | 0.5M Tris-HCl Buffer |
|  | 10% Sodium Dodecyl Sulfate Solution |
|  | 10% Ammonium Persulfate (APS) |
|  | N, N, N\', N\'-Tetramethylethylenediamine (TEMED) |
|  | ddH2O |

**Procedure:**

1. Mix 4 mL lower gel (2x) and 4 mL lower gel buffer (2x).
2. Add 80 μL gel accelerator and quickly add the liquid into the glass slit.
3. Use CH3-COH-CH3 to seal the lower gel.
4. Wait until the gel is completely set (6-10 minutes).
5. Mix 1 mL upper gel (2x) and 1 mL upper gel buffer (2x).
6. Add 20 μL gel accelerator and add the liquid into the glass slit until it is full.
7. Insert the sample comb and wait for 15 minutes.
8. When the upper gel is completely concrete, plug out the comb.

**3.2 Transformation into *E. coli* Origami 2(DE3) Expression Strain**

**Purpose:** To introduce the target plasmid into the expression strain for protein production.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Centrifuge |
|  | Incubator |
|  | Water bath pot |
|  | Ice-making machine |
| Chemicals and Reagents | pRSFDuet-PDI-Thaumatian A/B/C/D plasmid |
|  | *E. co*li Origami 2(DE3) competent cells |
|  | LB liquid medium (K⁺) |

**Procedure:**

1. Thaw competent cells on ice for 10 minutes.
2. Add 1-5 µL plasmid DNA to 50 µL cells, mix gently, then incubate on ice for 30 min.
3. Heat-shock at 42°C for 45 seconds, then immediately put on ice for 2 minutes.
4. Add 500 µL LB medium (no antibiotic), incubate at 37°C for 1 hour.
5. Plate 100 µL of the transformed cells on LB agar plates (antibiotic), and incubate at 37℃ for 12-16 hours.

**3.3 Ultrasonic Disruption**

**Purpose:** To break the cell apart by sonicating, and to remove cell debris and insoluble
components by centrifugation, obtaining a crude protein supernatant containing the target protein as the starting
material for subsequent affinity purification.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Centrifuge |
|  | Ultrasonic Cell Disruptor |
|  | Ice-making machine |
| Chemicals and Reagents | Bacterial pellet |
|  | Lysis buffer |

**Procedure:**

1. Place the bacterial solution on ice for 5 minutes to cool it down.
2. Setting parameters: 20kHz, amplitude 40%, pulse 5 seconds on/5 seconds off, total time 3-5 minutes.
3. Keep an ice bath during the process, and avoid the temperature exceeding 4℃.
4. A 10 μL sample was examined under a microscope (to check if the cells were lysed).
5. Centrifuge at 5000 rpm, 4 °C for 15 min, and collect the supernatant.

**3.4 Protein Purification**

**Purpose:** To selectively bind and purify His-tagged target proteins from the crude protein
supernatant using Ni-affinity chromatography, while including a control group to evaluate the efficiency and
specificity of the purification process.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Gravity column |
|  | Centrifuge tube |
| Chemicals and Reagents | Ni-NTA resin |
|  | Wash buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 40 mM imidazole) |
|  | Elution buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 250 mM imidazole) |

**Procedure:**

1) Transfer the crude protein supernatants into two micro-centrifuge tubes for each sample. Load 4 mL of crude
protein supernatant from one tube into an affinity chromatography column.

2) Add 1 mL of Ni solution to the affinity chromatography column, and collect the flow-through for later analysis.

3) Wash to remove nonspecific proteins by adding 1 mL of 25 mM imidazole solution, repeating the wash step five
times.

4) Elute the target protein by adding 250 μL of 500 mM imidazole solution, repeating the elution step twice.

**3.5 SDS-PAGE**

**Purpose:** To confirm protein size and purity.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Vertical electrophoresis system |
|  | Microcentrifuge |
|  | Water bath |
|  | Vortex mixer |
| Chemicals and Reagents | 12.5% SDS-PAGE gel |
|  | Running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) |
|  | Protein samples |
|  | Protein ladder |
|  | Coomassie Blue stain |
|  | Destaining solution |

**Procedure:**

1. Mix 10 µL protein sample with 2× Laemmli buffer in clean centrifuge tubes, Vortex for 10 seconds. Boil for 5
   minutes.
2. Load the ladder and samples into the lanes. Run at 120 V for 90 min.
3. Stain with Coomassie Blue stain for 30 minutes. Incubate the gel in destaining solution overnight (12-16 hours).

',
"children": []
},
{
"title": '4. Functional Verification',
"content": '

**4.1 Thin Layer Chromatography (TLC)**

**Purpose:** To qualitatively detect the activities of AGE and NAL.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Silica gel TLC plates |
| Chemicals and Reagents | Solvent system |
|  | Sample solution |
|  | Standards |
|  | Staining reagent |

**Procedure:**

1. Prepare the mobile phase (n-butanol: glacial acetic acid: ethanol: water = 211: 1) and saturate the chamber for
   15–30 minutes.
2. Spot 2 μL of the supernatant on a silica plate (about 1 cm from the bottom), dry briefly with a hair dryer.
3. Develop the TLC plate until the solvent front reaches 5 – 8 cm, and dry the plate.
4. Dip the plate in 5% sulfuric acid ethanol, dry and heat it at 130°C for 5 minutes.

**4.2 Enzyme-Linked Immunosorbent Assay (ELISA)**

**Purpose:** To produce respective enzymes using the three different plasmids that have been
constructed.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Shaking incubator |
|  | Pipettes |
|  | Autoclave |
|  | Microtubes |
|  | Vortex |
|  | ELISA plate |
| Chemicals and Reagents | **Standards** |
|  | Sample Diluent |
|  | Antibody |
|  | Washing Buffer |
|  | Substrate Solution |
|  | Stop Solution |

**Procedure:**

1. Add 50 μL of standard substances with different concentrations to each standard well.
2. For sample wells, first add 10 μL of the test sample, and then add 40 μL of sample diluent; add nothing to the
   blank wells.
3. Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each
   standard well and sample well. Seal the reaction wells with a plate sealer and incubate in a 37°C water bath or
   incubator for 60 minutes.
4. Discard the liquid, pat dry on absorbent paper. Fill each well with washing solution, let it stand for 1 minute,
   shake off the washing solution, and pat dry on absorbent paper. Repeat the plate washing 5 times (a plate washer
   can also be used).
5. Add 50 μL of substrate A and 50 μL of substrate B to each well, and incubate at 37°C in the dark for 15 minutes.
6. Add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within
   15 minutes.

**4.3** **High Performance Liquid Chromatography (HPLC)**

**Purpose:** To produce respective enzymes using the three different plasmids that have been
constructed.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | HPLC System |
|  | Chromatography Workstation |
|  | Pipette |
|  | HPLC Column |
|  | Injection Needles |
| Chemicals and Reagents | Mobile Phase Solvents (HPLC Grade) |
|  | Standard Substance |
|  | Sample Solvents |

**Procedure:**

1) Preparation of Sialic Acid Standard Solution: Weigh 20 mg of sialic acid standard powder and dissolve it in the
mobile phase. Dilute the solution to a final volume of 100 mL in a volumetric flask to obtain a standard stock
solution with a concentration of 0.2 mg/mL. Perform gradient dilution of this stock solution to prepare a series
of standard solutions with different concentrations. After filtration through a 0.22 μm filter membrane, transfer
the standard solutions to sample vials for subsequent use.

2) Sample Preparation: Take the sample to be tested and centrifuge it at 15,000 rpm and 4 °C for 15 minutes.
Collect the supernatant and filter it through a 0.22 μm cellulose acetate filter membrane. Transfer the filtered
supernatant into HPLC vials for detection.

3) HPLC Instrument Startup and Equilibration: Set the chromatographic conditions (mobile phase: 5 mmol/L H₂SO₄;
flow rate: 0.6 mL/min; RID detector temperature: 35°C; column temperature: 65°C). Pump the mobile phase to flush
the chromatographic column for 30 minutes until the baseline on the workstation stabilizes (fluctuation ≤ 0.1
mAU).

4) Detection of Standards and Samples: Inject the series of standard solutions into the injector in ascending
order of concentration. For each concentration, perform the detection in triplicate. Record the retention time and
peak area of the standard in the chromatogram. Record the chromatogram of the sample.

',
"children": []
},
] %}
{% block title %}Experiments{% endblock %}
{% block lead %}EXPERIMENTS{% endblock %}

{% extends "layout.html" %}
{% set info\_data = [
{
"title": 'Overview',
"content": '

NeuAc (N-acetylneuraminic acid) synthesis relies on the collaborative action of enzymes AGE and NAL, with two
reversible reactions: one between GlcNAc (N-acetylglucosamine) and ManNAc (N-acetylmannosamine), and the other
between ManNAc and NeuAc (Figure 1). This process requires their precise coordination to drive
substrate-to-product conversion. The project constructed plasmids (pETDuet-AGE-NAL, pET28a-AGE-Linker-NAL,
pET28a-Scaffold) via homologous recombination, which served as carriers for expressing AGE, NAL, and Scaffold.
SDS-PAGE validation confirmed the successful synthesis and purification of these proteins, including the
AGE-Linker-NAL fusion protein and components of the combined system. Additionally, functional tests (notably TLC,
ELISA, and HPLC) characterized the enzyme activities of AGE and NAL under varied reaction time and temperature.
Collectively, these efforts laid the foundation for understanding and optimizing the NeuAc synthesis pathway
mediated by AGE and NAL.

![C:/Users/LinSh/AppData/Local/Temp/wps.FYyUPZwps](https://static.igem.wiki/teams/5766/result/01.webp)

**Figure 1 The pathway to synthesize NeuAc by using AGE and NAL**

',
"children": []
},
{
"title": 'Plasmid construction',
"content": '

**1.1 PCR Amplification**

We constructed pETDuet-AGE-NAL, pET28a-AGE-Linker-NAL, and pET28a-Scaffold by using homologous recombination. The
AGE, NAL, Scaffold, and AGE-Linker-NAL sequences were amplified by PCR. Figure 2 shows the agarose gel
electrophoresis results of the PCR. We can find out that the DNA fragment of *AGE* was approximately 1262
bp in length, *NAL* was 987 bp, *Scaffold* was 775 bp, *AGE-Linker-NAL* was 2117 bp, which
were consistent with the predicted size. This indicated that the target gene was successfully amplified.

![pcr组图_画板 1](https://static.igem.wiki/teams/5766/result/02.webp)

**Figure 2 Agarose gel electrophoresis results of PCR**

**1.2 Plasmid linearization**

The pETDuet-1 plasmid was used as a vector to ligate the AGE and NAL, with a length of 7563 bp after ligating both
target genes to the plasmid. The pET28a plasmid was used as a vector to ligate Scaffold and AGE-Linker-NAL, with
lengths of 5995 bp and 7342 bp, respectively, after ligation. Figure 3 shows the bands of these plasmids after
double enzyme digestion, which were consistent with the size. These results indicate a
successful linearization of the pETDuet-1 and pET28a plasmid.

![双酶切组图_画板 1](https://static.igem.wiki/teams/5766/result/03.webp)

**Figure 3 The gel electrophoresis validation of the linearized plasmids**

**1.3 Homologous Recombination**

Prior to constructing the pETDuet-AGE-NAL plasmid, we first completed the construction of pETDuet-AGE. This
construction employed a homologous recombination approach. The colonies obtained after transformation are shown in
Figure 4, indicating successful recombination and the acquisition of positive clones. Subsequently, we selected a
single colony for colony PCR verification. Electrophoresis revealed a specific band at 1262 bp, matching the
expected size, preliminarily confirming that the AGE fragment had been correctly recombined into the pETDuet
vector. To further validate the construction accuracy, we extracted the recombinant plasmid and submitted it for
sequencing. Sequence alignment confirmed that the AGE gene was correctly inserted with a fully intact reading
frame, showing no frameshifts or mutations. This indicates the successful construction of the pETDuet-AGE plasmid,
laying the foundation for subsequent introduction of the NAL gene and further dual-enzyme co-expression studies.

**![AGE验证图_画板 1](https://static.igem.wiki/teams/5766/result/04.webp)**

**Figure 4 pETDuet-AGE Plasmid Verification Diagram**

Building upon the successful construction of the pETDuet-AGE plasmid, we further employed homologous recombination
to insert the NAL gene into the vector, thereby constructing the pETDuet-AGE-NAL recombinant plasmid. Clear single
colonies were visible on the transformation plates, indicating successful recombination. Randomly selected
colonies were subjected to PCR validation. Electrophoresis analysis of the amplified products revealed a specific
band at the expected size (approximately 2249 bp), preliminarily confirming the correct insertion of the NAL gene.
Subsequently, positive clone plasmids were extracted for sequencing analysis. The sequencing results fully matched
the expected sequence, confirming that both the AGE and NAL genes were correctly integrated into the pETDuet
vector with intact reading frames and no frameshift mutations. These results indicate the successful construction
of the pETDuet-AGE-NAL plasmid, providing a reliable vector for subsequent dual-enzyme co-expression and catalytic
function studies.

![AGE+NAL组图_画板 1_画板 1](https://static.igem.wiki/teams/5766/result/05.webp)

**Figure 5 pETDuet-AGE-NAL Plasmid Verification Diagram**

We further employed homologous recombination to sequentially assemble the AGE gene, Linker sequence, and NAL gene
into the pET28a vector, constructing the pET28a-AGE-Linker-NAL recombinant plasmid. Following the transformation,
uniformly distributed single colonies were observed on the agar plate, indicating successful recombinant
transformation. Several colonies were randomly selected for colony PCR verification. Electrophoresis results
showed a specific band at the expected size (approximately 2303 bp for the AGE-Linker-NAL fragment), preliminarily
confirming the correct insertion of the three-gene fusion into the vector. To further validate construction
accuracy, positive clone plasmids were extracted for sequencing analysis. Sequence results fully matched the
expected sequence, confirming that the AGE, Linker, and NAL elements were correctly integrated into the pET28a
vector as designed, with precise codon transitions and no frameshift mutations. These results confirm the
successful construction of the pET28a-AGE-Linker-NAL triple fusion expression vector, providing essential material
for subsequent studies investigating the regulatory role of the Linker sequence on the spatial conformation and
catalytic efficiency of the dual enzymes.

![A-L--N组图_画板 1](https://static.igem.wiki/teams/5766/result/06.webp)

**Figure 6 pET28a-AGE-Linker-NAL Plasmid Verification Diagram**

We proceeded to employ homologous recombination to clone the artificial scaffold protein gene into the pET28a
vector, constructing the pET28a-Scaffold recombinant plasmid. Clear single colonies were observed on the plate
post-transformation, indicating successful transformation. Subsequently, multiple colonies were randomly selected
for colony PCR validation. Electrophoresis results showed a specific band at the expected size (approximately 755
bp), providing preliminary evidence that the Scaffold gene had been successfully inserted into the vector. To
further confirm construction accuracy, we extracted positive clone plasmids for sequencing analysis. The
sequencing results fully matched the expected sequence, confirming that the Scaffold gene was correctly integrated
into the pET28a vector with an intact coding frame, free of frameshifts or mutations. These results indicate the
successful construction of the pET28a-Scaffold plasmid, laying the foundation for subsequent studies investigating
the regulatory role of scaffold proteins in multi-enzyme assembly and metabolic pathway efficiency.

![支架组图_画板 1](https://static.igem.wiki/teams/5766/result/07.webp)

**Figure 7 pET28a-Scaffold Plasmid Verification Diagram**

',
"children": []
},
{
"title": 'Protein expression',
"content": '

**2.1 Heat shock conversion into BL21(DE3)**

Each of the recombinant plasmids was independently transformed into *E. coli* BL21(DE3) competent cells
using the heat shock method. The competent cells were first incubated with plasmid DNA on ice and then exposed to
a brief heat shock at 42 ℃ to facilitate DNA uptake. Following recovery in LB medium, the cells were plated on
selective LB agar plates containing the corresponding antibiotic and incubated overnight at 37 ℃.

Co-transform pET28a-Scaffold plasmid and pETDuet-AGE-NAL plasmid into E. coli BL21(DE3) competent cells.
Simultaneously, individually transform pET28a-AGE-Linker-NAL and pETDuet-AGE-NAL single plasmids into the same
host strain. The co-transformed colonies grew on plates containing both ampicillin and kanamycin, while the
remaining colonies grew on ampicillin-only plates. Clear, single colonies were observed on all plates
post-transformation, indicating successful transformation for each combination.

![](https://static.igem.wiki/teams/5766/result/08.webp)

**Figure 8 Agar plate showing BL21 colonies after transformation**

**Notes: A: pETDuet-AGE-NAL, B: pET28a-AGE-Linker-NAL, C: pET28a-Scafford + pETDuet-AGE-NAL.**

Single colonies from each experimental group were selected for colony PCR verification. Results showed: The total
transformation group simultaneously amplified the Scaffold characteristic band (approximately 755 bp) and the
AGE-NAL fusion fragment band (approximately 2249 bp); The pET28a-AGE-Linker-NAL group amplified the expected-size
Linker-AGE-NAL band (approximately 2303 bp), while the pETDuet-AGE-NAL group exhibited the characteristic AGE-NAL
band (approximately 2249 bp). Electrophoresis results for all conditions aligned with expectations. These findings
confirm that all three transformation combinations successfully yielded positive clones, establishing a reliable
experimental foundation for subsequent comparisons of functional differences between the scaffold-mediated
multienzyme assembly system and the direct fusion expression system.

![BL21菌落PCR 2](https://static.igem.wiki/teams/5766/result/09.webp)

**Figure 9 PCR Identification of Colonies from Three Plasmids**

**2.2 Lysate protein concentration**

Each PCR-verified colony was inoculated into liquid LB medium and cultured overnight, followed by scale-up
fermentation. After cell harvest via centrifugation, bacterial pellets were subjected to ultrasonic lysis to
release intracellular contents.

**2.3 SDS-PAGE verification**

After protein purification using a Nickel column, SDS-PAGE analysis was performed on both crude and purified
samples. Proteins were separated on a 15% polyacrylamide gel and stained with Coomassie Brilliant Blue.

For pET28a-AGE-Linker-NAL, as shown in Figure 10, a band corresponding to AGE-Linker-NAL was detected in the
elution solution (band 5), confirming the successful expression of this fusion protein and its effective
separation and identification after purification.

![](https://static.igem.wiki/teams/5766/result/10.webp)

**Figure 10 SDS-PAGE for AGE-Linker-NAL**

For pETDuet-AGE-NAL, Figure 11 showed that clear bands corresponding to AGE and NAL (at their expected positions)
were observed in the elution solutions 1 and 2 (bands 5, 6), indicating the successful expression of both proteins
and their effective enrichment after purification.

![](https://static.igem.wiki/teams/5766/result/11.webp)

**Figure 11 SDS-PAGE for AGE and NAL**

Additionally, for the co-expression of pET28a-Scaffold and pETDuet-AGE-NAL, Figure 12 also revealed that in the
elution solution 1 and 2 (bands 5, 6), not only bands of AGE and NAL but also a visible band corresponding to
Scaffold were present, demonstrating that all proteins in the Scaffold + AGE + NAL fusion system were expressed
and purified.

![](https://static.igem.wiki/teams/5766/result/12.webp)

**Figure 12 SDS-PAGE for Scaffold, AGE, and NAL**

',
"children": []
},
{
"title": 'Functional Test',
"content": '

**3.1 Thin-Layer Chromatography (TLC)**

The activities of AGE and NAL were qualitatively analyzed using TLC at two time points: 30 and 60 minutes. As
shown in Figure 13, in the AGE-catalyzed reactions, the band corresponding to the non-denatured enzyme at 30
minutes migrated similarly to the product reference, while the denatured enzyme at 60 minutes co-migrated with the
substrate. This indicates that 30 minutes was sufficient for optimal AGE activity under these conditions. For NAL,
the TLC results showed separation primarily at the substrate positions, suggesting that the current mobile phase
may not be ideal for resolving NAL-derived products due to their high polarity.

![](https://static.igem.wiki/teams/5766/result/13.webp)

**Figure 13 TLC for AGE and NAL under 30 and 60 minutes**

To further validate the generation of ManNAc and NeuAc, we optimized the developing solvent system for thin-layer
chromatography (TLC). After reacting at 42°C for 30 minutes, samples underwent two duplicate developments using
freshly prepared developing solvent, with TLC plates thoroughly dried using a hair dryer after each development.
Subsequently, TLC plates were immersed in a developing agent and heated at 130°C for 5 minutes for visualization.

Results showed that in the three distinct enzyme structures—AGE-NAL (A-N), AGE-Linker-NAL (A-L-N), and Scaffold +
NAL-AGE (S+A-N)—no bands corresponding to ManNAc or NeuAc were detected in the A-N group. In contrast, both A-L-N
and S+A-N groups exhibited distinct target product bands. This result indicates that introducing Linker or
Scaffold modules into the enzyme structure effectively prevents reversible reactions during metabolism, thereby
enhancing the synthetic efficiency of ManNAc and NeuAc.

![AGE+NAL组图_画板 1](https://static.igem.wiki/teams/5766/result/14.webp)

**Figure 14 TLC for AGE and NAL under 30 minutes**

We conducted systematic TLC analysis of the enzymatic activities of three enzyme constructs—AGE-NAL (A-N),
AGE-Linker-NAL (A-L-N), and Scaffold+NAL-AGE (S+A-N)—reacted for one hour at three temperature conditions: 25°C,
37°C, and 42°C. As shown in Figure 15A, the A-N construct yielded the highest amount of ManNAc in the 1-hour
incubation group, with no detectable NeuAc product formation at this time point. This indicates that A-N tends to
accumulate the intermediate ManNAc over shorter reaction times.

When incubation was extended to 2 hours, as shown in Figure 15B, both the A-L-N and S+A-N constructs exhibited
distinct NeuAc bands whose intensity approached that of the target product reference band. This indicates that
these constructs efficiently catalyze NeuAc synthesis upon extended reaction time. Compared to the 1-hour
incubation group, the 2-hour incubation group exhibited more pronounced product bands at 37°C, indicating that
appropriately extending the reaction time significantly enhances catalytic efficiency.

Collectively, these results indicate that the A-N construct favors ManNAc accumulation in short-duration
reactions, while the A-L-N and S+A-N constructs promote final NeuAc production after extended reaction times, with
37°C and 2 hours representing optimal conditions. This phenomenon indicates significant differences in catalytic
properties among different enzyme constructs. The introduction of linkers or scaffolds may promote the conversion
of reaction intermediates into end products, thereby enhancing overall catalytic efficiency.

**![全细胞催化组图_画板 1](https://static.igem.wiki/teams/5766/result/15.webp)**

**Figure 15 TLC for NAL-AGE (A-N), AGE-Linker-NAL (A-L-N), and Scaffold + NAL-AGE (S+A-N) under 25°C, 37°C, and
42°C for 1 hour,and 2 h**

**3.2 Enzyme-linked immunosorbent assay (ELISA)**

To evaluate the catalytic performance of the dual-enzyme system, an ELISA-based quantification assay was
conducted.

**Table 1 The standard table of Neu5Ac**

|  |  |
| --- | --- |
| Concentration (ng/mL) | Absorbance |
| 0 | 0.0889 |
| 10 | 0.5624 |
| 20 | 1.0481 |
| 40 | 1.5943 |
| 80 | 2.0665 |
| 160 | 2.3355 |

A 4-parameter logistic regression (4PL) model was employed for standard curve fitting, with the equation shown in
Figure 16, which exhibited a high correlation coefficient (R² = 0.9999).

![](https://static.igem.wiki/teams/5766/result/16.webp)

**Figure 16 The equation and the standard curve for the ELISA experiment**

The absorbance measured in the experiment was substituted into the equation for calculation to obtain the initial
detected concentration of sialic acid. The actual content of sialic acid in the samples was then derived by
multiplying by the dilution factor (5 times), as shown in Table 2.

**Table 2**

|  |  |  |  |
| --- | --- | --- | --- |
| Samples | Absorbance | Initial detection concentration | Actual concentration |
| Control Group | 0.0457 | 0.0000 | 0.0000 |
| A-N 1 mg/mL | 1.6396 | 41.9429 | 209.7146 |
| A-N 5 mg/mL | 0.2220 | 3.4540 | 17.2700 |
| A-N 10 mg/mL | 0.4331 | 7.4243 | 37.1216 |
| A-L-N 1 mg/mL | 0.6802 | 12.1123 | 60.5615 |
| A-L-N 5 mg/mL | 0.7649 | 13.8322 | 69.1611 |
| A-L-N 10 mg/mL | 1.0244 | 19.7337 | 98.6683 |
| S+A-N 1 mg/mL | 1.2331 | 25.5186 | 127.5932 |
| S+A-N 5 mg/mL | 0.8622 | 15.9175 | 79.5877 |
| S+A-N 10 mg/mL | 1.4269 | 32.2130 | 161.0652 |

The final results were plotted in Figure 17. For the enzyme construct A-N, the sialic acid synthesis efficiency
was the highest at a concentration of 1 mg/mL, and decreased at medium to high concentrations (5-10 mg/mL). At a
low concentration, the rate of the first-step reaction in sialic acid synthesis was slightly higher than that of
the second-step reaction, resulting in the overall reaction proceeding toward sialic acid synthesis. Under high
concentration conditions, the product sialic acid was decomposed into intermediate products by the NAL enzyme,
which were further decomposed by the AGE enzyme.

For the enzyme construct A-L-N, the content of sialic acid showed an upward trend as the concentration increased.
Compared with the maximum value of 1 mg/mL in the A-N group, the detection values at various concentrations in the
A-L-N group were relatively stable. This indicated that the role of the Linker is to stabilize enzyme function
rather than overactivate it.

For the enzyme construct S+A-N, all detection indicators were generally higher than those in the A-L-N group. At
the concentration of 10 mg/mL, the detected value was second only to that of the A-N group at 1 mg/mL. Through the
clustering effect, the Scaffold formed high-concentration A-N enzyme clusters locally, significantly improving the
reaction efficiency. Meanwhile, by binding to the AGE and NAL enzymes, the Scaffold induced their conversion from
an inactive conformation to an active conformation, which could directly enhance the catalytic efficiency of
individual enzymes. This was the core reason why the detected values of the S+A-N group were higher than those of
the A-L-N group.

**In conclusion, the enzyme construction Scaffold+AGE-NAL can maximize the efficiency of sialic acid synthesis at
medium to high concentrations, making it the optimal solution for efficiently increasing sialic acid
production.**

![](https://static.igem.wiki/teams/5766/result/17.webp)

**Figure 17 Concentration of Neu5Ac tested by the ELISA Kit calculated by the standard curve**

**3.3 High-Performance Liquid Chromatography (HPLC)**

The catalytic experiment of the A-N enzyme construct was illustrated in Figure 18A. The sialic acid content
reached its highest level at a reaction time of 60 minutes across all three reaction temperatures. As the reaction
time prolonged, the sialic acid yield exhibited a decreasing trend. We hypothesize that this phenomenon was
associated with the reversibility of the catalytic reaction. When the content of the intermediate product
ManNAc-6P accumulated to a certain extent, the reaction proceeded in the reverse direction. Consequently, the
substrate for the second step of the reaction was reduced, leading to a decrease in the final yield of sialic
acid. Under the condition of a reaction time of 60 minutes, the reaction temperature of 42 °C yielded the highest
accumulation of sialic acid. Overall, the optimal reaction system for enzyme construct A-N is as follows:
temperature 42 °C + reaction time 60 minutes.

The similar results were also observed when enzyme constructs A-L-N and S+A-N were used, as shown in Figure 18B
and 18C. Notably, when the S+A-N enzyme construct was used, the accumulation of sialic acid rebounded under the
conditions of 42 °C and a reaction time of 240 minutes. This may be attributed to the fact that the protein
scaffold effectively reduced the occurrence of reversible reactions under such reaction conditions. Overall, the
optimal reaction systems for both enzyme constructs A-L-N and S+A-N were as follows: temperature 42 °C + reaction
time 60 minutes.

![](https://static.igem.wiki/teams/5766/result/18.webp)

**Figure 18 Sialic acid production by each enzyme construct under different reaction times and
temperatures**

The sialic acid production of the three enzyme constructs under the condition of 42 °C, 240 minutes was
illustrated in Figure 19. Although the sialic acid content decreased as the reaction time prolonged, the enzyme
construct S+A-N exhibited the smallest degree of decrease and achieved the highest final accumulated product
concentration.
**This indicated that construct S+A-N can effectively reduce the occurrence of reversible reactions and was thus
the optimal enzyme construct for producing sialic acid.**

![](https://static.igem.wiki/teams/5766/result/19.webp)

**Figure 19 Sialic acid production by three enzyme constructs under 42 ℃, 240 minutes**

Through detailed analysis of the HPLC peak chart in Figure 20, the successful generation of sialic acid and the
significant catalytic advantage of the S+A-N construct can be further confirmed.

In the chromatogram of the S+A-N construct, a distinct and sharp peak appears at approximately 11 minutes
retention time, which aligns perfectly with the characteristic peak of sialic acid in the standard sample. This
peak exhibits symmetry and a large area, indicating high product concentration and excellent separation with no
significant tailing.

In contrast, although product peaks are detectable at the same retention time for other constructs (e.g., A-N and
A-L-N), their response intensity is significantly lower than that of S+A-N. These peaks are smaller and
accompanied by minor impurity peaks. Particularly in the A-N sample, a chromatographic peak belonging to the
intermediate ManNAc (retention time approximately 3 minutes) appeared before the target product peak, further
indicating incomplete reaction and lower catalytic efficiency.

In summary, the HPLC quantitative results align with previous activity assessment conclusions: the S+A-N construct
most effectively catalyzes sialic acid synthesis, yielding the highest product yield and optimal purity, fully
demonstrating its superiority as a highly efficient catalytic module.

![](https://static.igem.wiki/teams/5766/result/20.webp)

**Figure 20 Chromatograms of sialic acid content under 42 ℃, 240 minutes**

',
"children": []
},
{
"title": 'Future expectation for experiment',
"content": '

Our experimental data have clearly demonstrated the successful enzymatic synthesis of sialic acid, confirming the
catalytic functionality of the enzyme constructs under the applied conditions. Nevertheless, several aspects of
the methodology offer opportunities for further refinement that would enhance the robustness and interpretability
of the results. For instance, the developing solvent system and coloring reagents used in the thin-layer
chromatography (TLC) assays could be systematically optimized to improve resolution between substrates and
products, thereby allowing more accurate qualitative and quantitative analysis. In addition, future studies would
benefit from more comprehensive functional assessments of the enzyme constructs—such as kinetic studies, stability
tests under various conditions, and structural analyses—to better understand their catalytic mechanisms and
performance limitations. Implementing these improvements would not only strengthen the validity of our current
conclusions but also provide deeper insights into the enzymatic synthesis process, further establishing a reliable
foundation for subsequent research and potential applications.

',
"children": []
},
] %}
{% block title %}Results{% endblock %}
{% block lead %}RESULTS{% endblock %}