iGEM 2025 — Team depu

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"title": '1. Cycle 1 Lipase selection',
"content": '
',
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{
"title": '2. Cycle 2 Promoter and signal peptide optimization',
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{
"title": '3. Cycle 3 Final plasmid construction and test',
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{% block title %}Notebook{% endblock %}
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![](https://static.igem.wiki/teams/5790/common/notebook.webp)
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{
"title": 'Overview',
"content": '

To address the global challenge of waste oil recycling and support the enzymatic production of biodiesel—an environmentally
friendly alternative to conventional diesel—we engineered the yeast *Pichia pastoris* (*P. pastoris.*) as a robust cellular
factory for high-level production of engineered lipases with enhanced tolerance and catalytic activity. Recognizing that
natural microbial lipases often lack the stability and efficiency required for industrial-scale conversion, we employed a
systematic protein and genetic engineering approach to optimize three key elements: lipase, promoter, and signal peptide.
After selecting the best part, we combined them into a complete lipase production and secretion system, thereby obtaining a
strain with the highest expression level and enzyme activity, which helps to achieve efficient production of lipase and lays
a solid foundation for its application in biodiesel conversion.

In this project, we carried out a total of three DBTL cycles (**Figure 1**):

**Cycle 1:** Screening and Testing of Lipase

**Cycle 2:** Optimization and Testing of Control Components

* Cycle 2a: Promoter Optimization
* Cycle 2b: Signal peptide Optimization

**Cycle 3:** Building and Testing the Optimal Combination

![](https://static.igem.wiki/teams/5790/engineer/1.webp)

**Figure 1** The design concept of the project. (A) Technical route, (B) DBTL cycles.

',
"children": []
},
{
"title": 'Cycle 1: Screening and Testing of Lipase',
"content": '',
"children": [
{
"title": 'Design 1',
"content": '

Firstly, three promising lipases—CALB (from *Candida antarctica*), GGL (from *Galactomyces geotrichum Y05*), and LipZ01 (from oil-contaminated soil)—were selected for their distinct advantages in activity and stability (**Table 1**).

**Table 1** Three lipase used in this project.

|  |  |  |  |
| --- | --- | --- | --- |
| Name | Source | Genbank | Description |
| CALB | Candida antarctica | [Z30645.1](https://www.ncbi.nlm.nih.gov/nuccore/Z30645.1#tdsub) | One of the most widely studied and applied lipases due to its extensive biocatalytic applications [1]. |
| GGL | Galactomyces geotrichum Y05 | [DQ841279.1](https://www.ncbi.nlm.nih.gov/nuccore/DQ841279.1#tdsub) | Excellent heat resistance and tolerance to organic solvents [2]. |
| LipZ01 | Soil-derived metagenome | [AY304500](https://www.ncbi.nlm.nih.gov/protein/32364181#tdsub) | Good stability over a wide range of temperatures and under alkaline reaction conditions [3]. |

After the company synthesized three lipases that had been optimized for codons, we inserted them into a *P. pastoris* expression vector pPICZαA, which includes the methanol-inducible AOX1 promoter, the α-factor secretion signal, and
a C-terminal His-tag, followed by the AOX1 terminator. The Zeocin resistance gene enables selection in transformants
(**Figure 2**).

![](https://static.igem.wiki/teams/5790/engineer/2.webp)

**Figure 2** Plasmid Design diagram in cycle 1.

',
},
{
"title": 'Build 1',
"content": '

We firstly amplified the pPICZαA and three lipase gene, followed by agarose gel electrophoresis. The results showed the
successful amplification of pPICZαA vector (3476 bp), CALB (954 bp), GGL (1692 bp), and LipZ01 (1428 bp)—with bands
corresponding to their expected sizes under UV illumination (**Figure 3**).

![](https://static.igem.wiki/teams/5790/engineer/3.webp)

**Figure 3** The amplification results of the DNA fragments.

Following the amplification of the lipase genes and plasmid vector, the transformation plates confirmed efficient plasmid
introduction into *E. coli* DH5α competent cell (**Figure 4A**). Colony PCR analysis further validated
the presence of the correct insert sizes, with distinct bands corresponding to CALB (1443 bp), GGL (2179 bp), and LipZ01
(1912 bp) observed across multiple clones (**Figure 4B**). Sequencing results confirmed the precise integration
of each expression cassette (**Figure 4C**). These results collectively demonstrate the accurate assembly of
all three plasmids.

![](https://static.igem.wiki/teams/5790/engineer/4.webp)

**Figure 4** Construction result of three plasmids.

(A) Transformation plates. (B) Colony PCR result. (C)Sequencing result.

The recombinant plasmids pPICZαA-CALB, pPICZαA-GGL, and pPICZαA-LipZ01 were successfully extracted and linearized by
*SacI* enzyme prior to transformation, as confirmed by agarose gel electrophoresis (**Figure 5A**).
Linearizing the plasmid is a critical step because it exposes homologous ends that facilitate targeted integration into the
*P. pastoris* genome via homologous recombination, significantly increasing transformation efficiency compared to
circular plasmids.

Next, we used the lithium chloride transformation method to transfer the linearized plasmid into *P. pastoris*. This
method can generate active yeast cells with permeable cell membranes, enabling the linearized plasmid to smoothly enter the
cells. For the pPICZαA vector, the DNA that has been linearized will specifically integrate into the yeast genome through
homologous recombination at the AOX1 locus in the yeast genome. This process ensures that the target gene can be stably
expressed under the regulation of the inducible AOX1 promoter.

As shown in **Figure 5B,** the linearized plasmids were individually transformed into *P. pastoris* strains, with successful transformants growing on selective YPD plate. The yeast colony PCR results further verified the
correct genomic integration of each lipase gene, showing specific amplification products of 954 bp (CALB), 1692 bp (GGL),
and 1428 bp (LipZ01) (**Figure 5C**). These results collectively demonstrate the successful construction of
three recombinant *P. pastoris* strains capable of expressing the target lipases, providing a solid foundation
for subsequent protein expression and functional studies.

![](https://static.igem.wiki/teams/5790/engineer/5.webp)

**Figure 5** Construction result of *P. pastoris* containing lipase.

(A) Plasmid Linearization result. (B) Transformation plates. (C) Yeast colony PCR result.

',
},
{
"title": 'Test 1',
"content": '

* **Lipase secretion and purification**

Subsequently, we inoculated the yeast transformants containing the *CALB* gene and activated them in the BMGY medium.
Then, using the BMMY medium, we began to induce the secretion of CALB. **Figure 6** presented the SDS-PAGE
analysis of recombinant lipase secretion in *P. pastoris* after 72-hour fermentation. A distinct protein
band at approximately 44 kDa, corresponding to the expected molecular weight of CALB (43.8 kDa), was clearly observed in the
culture supernatant of all three tested strains at 48 h and 72 h, confirming successful extracellular secretion (**Figure 6A)**. In contrast, no visible bands were detected at the predicted molecular weights for GGL (63.1 kDa) or LipZ01 (51.1 kDa) in
any strain or time point (**Figure 6B-C)**.

The successful secretion of CALB can be attributed to its compatibility with the α-factor secretion signal and efficient
folding in the secretory pathway. The absence of GGL and LipZ01 bands suggests potential issues such as inefficient signal
peptide recognition, protein misfolding leading to endoplasmic reticulum retention, or intracellular degradation. These
results indicate that while the expression system supports CALB secretion, further optimization of the secretion signal or
co-expression of chaperones may be necessary for efficient production of GGL and LipZ01.

![](https://static.igem.wiki/teams/5790/engineer/6.webp)

**Figure 6** SDS-PAGE results of (A) CALB, (B)GGL, and (C) LipZ01 secretion.

We found that the purity of the lipase secreted into the supernatant was not high enough. To avoid affecting the subsequent
quantitative analysis of the lipase and the enzyme activity test, we carried out nickel column purification on it. Since the
crude supernatant contained impurities that could interfere with accurate quantification and activity assays, we
concentrated it using an ultrafiltration device and exchanged the buffer to PBS (pH 7.4) to optimize binding efficiency.
Following washing and elution steps, a distinct band at approximately 44 kDa, corresponding to CALB, was clearly observed in
the elution fractions (**Figure 7**, E1 and E2), confirming successful purification of the target protein to
high purity. In contrast, no specific bands were detected at the expected molecular weights for GGL (63.1 kDa) or LipZ01
(51.1 kDa) in their respective elution lanes, despite applying the same concentration and purification procedure. This
result is consistent with the earlier secretion data and further confirms that these two lipases were not successfully
secreted in a soluble, His-tagged form, or were present at levels below the detection limit.

![](https://static.igem.wiki/teams/5790/engineer/7.webp)

**Figure 7** Purification results of three lipase.

* **Quantitative analysis of lipase**

Next, we use the BCA method to determine the concentration of the lipase. The BCA method relies on the reduction of Cu²⁺ to
Cu⁺ by peptide bonds in an alkaline medium, with the resulting Cu⁺ ions forming a purple complex with BCA that can be
measured at 562 nm. A series of bovine serum albumin (BSA) standards (25-500 μg/mL) were used to establish a linear
calibration curve, which exhibited excellent linearity with the equation Y = 0.0009982\*X + 0.1326 and a coefficient of
determination (R²) of 0.9934 (**Figure 8**). The high R² value indicateed a strong correlation between
absorbance and protein concentration, validating the reliability of this standard curve for subsequent quantification of
lipase samples.

![](https://static.igem.wiki/teams/5790/engineer/8.webp)

**Figure 8** The BSA standard curve results of the BCA method

This standardized protocol was applied to determine the concentration of purified lipases in the culture supernatant,
enabling accurate comparison of expression levels among CALB, GGL, and LipZ01 under different experimental conditions. The
BCA assay results quantitatively confirmed the successful secretion of recombinant CALB, which achieved a concentration of
68.21 μg/mL, significantly higher than the negative control (0.96 μg/mL). In contrast, GGL and LipZ01 showed only minimal
concentrations (4.89 and 1.61 μg/mL, respectively), consistent with earlier SDS-PAGE and purification data indicating their
secretion failure (**Figure 9)**. These results validated the effectiveness of the expression and purification
system for CALB, while underscoring the need for further optimization to enable detectable secretion of GGL and LipZ01.

![](https://static.igem.wiki/teams/5790/engineer/9.webp)

**Figure 9** The quantitative results of the concentration of purified lipase.

* **Enzyme activity test of lipase**

We determined the activity of the lipase using the p-nitrophenol method. This assay is based on the hydrolysis of
p-Nitrophenyl esters by lipase, releasing yellow-colored 4-Nitrophenol (4-NP) that absorbs at 450 nm. A series of 4-NP
standards (0–0.1 mmol/L) were prepared, and their absorbance values were measured to establish a linear calibration curve.
The fitted equation Y = 5.924x + 0.01800 exhibited excellent linearity (R² = 0.9857), confirming a strong correlation
between absorbance and 4-NP concentration (**Figure 10)**. This reliable standard curve enables accurate
quantification of lipase activity in subsequent enzymatic assays.

![](https://static.igem.wiki/teams/5790/engineer/10.webp)

**Figure 10** Standard curve of 4-Nitrophenol

Finally, the lipase activity of the three recombinant strains was measured. The CALB-expressing strain demonstrated the
highest enzymatic activity at 28.59 ± 0.37 U/mL, which was approximately 3.5-fold higher than the activities of GGL (8.15 ±
0.14 U/mL) and LipZ01 (8.41 ± 0.09 U/mL). Statistical analysis (​**​​**​p < 0.0001) confirmed that CALB
activity was significantly higher than both GGL and LipZ01, while no significant difference (ns) was observed between GGL
and LipZ01. The CALB-expressing strain showed significantly higher activity than the negative control (pPICZαA, 5.99 ± 0.70
U/mL), validating the successful expression of it (**Figure 11)**. These results indicate that CALB exhibits
superior catalytic efficiency under the tested conditions.

![](https://static.igem.wiki/teams/5790/engineer/11.webp)

**Figure 11** Lipase activity test result.

',
},
{
"title": 'Learn 1',
"content": '

During this round of DBTL cycle, we found that CLAB was able to successfully be secreted from the cells of
*P. pastoris* into the supernatant of the culture medium. The enzymatic activity revealed that only CALB was
successfully secreted extracellularly, demonstrating measurable enzymatic activity (reaching 28.59 ± 0.37 U/mL). Therefore,
in the subsequent optimization phase, we will focus on CALB, specifically targeting the enhancement of its expression and
secretion efficiency through promoter and signal peptide optimization.

However, GGL and LipZ01 did not show secretion. Through literature research, we summarized the possible reasons and
solutions, and can optimize this expression system in future studies (**Table 2**).

**Table 2** Reasons and solutions for the failure of GGL and LipZ01 secretion.

|  |  |
| --- | --- |
| Reason | Solution |
| 1. The molecular weights of GGL and LipZ01 are relatively large (50-63 kDa), making them prone to aggregation or misfolding in the endoplasmic reticulum and making it difficult for them to enter the secretion pathway [4]. | They can be assisted in folding by co-expressing molecular chaperones, such as endoplasmic reticulum oxidation 1(Ero1) and protein disulfide isomerase (PDI), or by changing the expression system [5-6]. |
| 2. The α-factor signal peptide we use may be incompatible with GGL and LipZ01, preventing the protein precursor from being effectively recognized and processed, and thus unable to be properly translocated to the endoplasmic reticulum [7]. | By replacing or modifying the signal peptide, we can enhance its compatibility with the target protein and the host secretion mechanism [7]. |
| 3. The *P. pastoris* strain GS115 Mut+ used in this project may have significant differences in expression and secretion capabilities for different lipases. | We can try replacing it with other strains (such as X33 or KM71H) [8]. |

',
},
]
},
{
"title": 'Cycle 2: Optimization and Testing of Control Components',
"content": '',
"children": []
},
{
"title": 'Cycle 2a: Promoter Optimization',
"content": '',
"children": [
{
"title": 'Design 2a',
"content": '

Achieving efficient expression of lipases in *P. pastoris* requires careful selection and optimization of promoters,
as they directly govern the efficiency and intensity of target gene transcription initiation [9]. The pPIC9K vector is a
commonly used tool in the *P. pastoris* expression system, which incorporates the potent methanol-inducible AOX1
promoter, and utilizes the α-factor signal peptide to direct the secretory expression of recombinant proteins. This vector
is marked for prokaryotic screening in *E. coli* with ampicillin (Amp) and kanamycin (Kan) resistance, while
screening in *P. pastoris* utilizes the HIS4 marker. (**Figure 12)**.

![](https://static.igem.wiki/teams/5790/engineer/12.webp)

**Figure 12** Plasmid Design diagram in cycle 2a.

To systematically evaluate the impact of different promoters on lipase expression levels, we replaced the original AOX1
promoter with three candidate promoters—AOX713, FDH1, and FLD1 (**Table 3**). Through this strategy, we aimed
to identify the optimal promoter capable of maximizing lipase transcription levels and final yield, laying the foundation
for constructing an efficient *P. pastoris.* cell factory.

**Table 3** Promoters and signal peptides used in this project.

|  |  |  |
| --- | --- | --- |
| Name | Source | Description |
| AOX1  (Control) | Endogenous promoter of *P. pastoris* | The AOX1 promoter is the most widely used methanol-inducible promoter in *P. pastoris* [10]. |
| AOX713 | Rational Design of AOX1 Promoter | Designed by rationally removing inhibitory elements and replicating activating elements [11]. |
| FDH1 | Formate dehydrogenase promoter from *P. pastoris* | This classic NAD⁺-specific FDH catalyzes formate oxidation coupled with NAD⁺ reduction to NADH [12]. |
| FLD1 | Formaldehyde dehydrogenase 1 promoter from *P. pastoris* | Methanol or methylamine induction achieves AOX1-promoter-comparable expression levels [13]. |

',
},
{
"title": 'Build 2a',
"content": '

Firstly, we performed PCR amplification on each promoter, the *CALB* gene, and the plasmid backbone. The results of agarose
gel electrophoresis showed that all target fragments were successfully amplified. The promoter fragments (AOX713, FDH1,
FLD1) and the Kan resistance gene fragment all presented clear bands, with sizes consistent with expectations (approximately
4366 bp, 4244 bp, and 4699 bp), and the *CALB* gene fragment (996 bp) was also successfully amplified (**Figure 13**). After gel excision, these fragments were used for the subsequent construction of the recombinant vector.

![](https://static.igem.wiki/teams/5790/engineer/13.webp)

**Figure 13** The amplification results of the DNA fragments.

Following the amplification of the different promoters, *CALB* gene, and plasmid vector, we combined them through
homologous recombination. the transformation plates confirmed efficient plasmid introduction into *E. coli* DH5α
competent cell. Colony PCR analysis demonstrated distinct bands of expected sizes: 2188 bp for AOX713-CALB, 2154 bp for
FDH1-CALB, and 2169 bp for FLD1-CALB. Sequencing results further verified the precise integration of each expression
cassette with correct promoter-signal peptide-CALB organization. These results collectively confirmed the accurate assembly
of three plasmid constructs, providing a reliable foundation for subsequent yeast transformation and protein expression
studies (**Figure 14**).

![](https://static.igem.wiki/teams/5790/engineer/14.webp)

**Figure 14** The colony PCR and sequencing results of plasmids.

The recombinant plasmids were then successfully extracted and linearized by *SalI* enzyme before transformation, as
confirmed by agarose gel electrophoresis. Each plasmid displayed a single, clear band after single enzyme digestion,
corresponding to the expected size of the linearized vector (**Figure 15A**). After enzyme digestion, the
linearized plasmids were transformed into *P. pastoris* competent cell, respectively.
**Figure 15B** showed that transformants of *P. pastoris* growing on selective plates for six
different constructs. Further colony PCR analysis confirmed genomic integration, with distinct bands observed at the
expected size of 954 bp for all constructs when compared to the DNA ladder (**Figure 15C**). These results
demonstrated the successful integration of the *CALB* gene expression cassettes into the yeast genome, validating the
effectiveness of the transformation procedure and providing a reliable foundation for subsequent protein expression studies.

![](https://static.igem.wiki/teams/5790/engineer/15.webp)

**Figure 15** Construction result of *P. pastoris* containing lipase.

(A) The linearization results of the plasmids. (B) Transformation plates. (C) Yeast colony PCR results.

',
},
{
"title": 'Test 2a',
"content": '

* **Lipase secretion and quantitative analysis**

Next, we conducted fermentation on each of these three strains. The SDS-PAGE analysis revealed distinct protein bands at
approximately 43.8 kDa in the 72-hour samples, corresponding to the expected molecular weight of CALB, while no such bands
were observed at 0 hours or in the negative control (pPIC9K) (**Figure 16A**). Quantitative analysis using the
BCA method demonstrated significant differences in CALB secretion levels among the promoters. The FDH1 promoter yielded the
highest concentration (93.82 ± 4.16 μg/mL), followed by AOX713 (86.04 ± 8.53 μg/mL), AOX1 (68.21 ± 4.94 μg/mL), and FLD1
(61.25 ± 3.32 μg/mL). Statistical analysis indicated that FDH1-driven expression was significantly higher than both AOX1 and
FLD1 (​​P ≤ 0.01) (**Figure 16B**).

![](https://static.igem.wiki/teams/5790/engineer/16.webp)

**Figure 16** (A)SDS-PAGE verification and (B) quantification of CALB under different promoter regulation.

* **Enzyme activity test of lipase**

Finally, we determined the lipase activity of the recombinant *P. pastoris* strains expressing CALB under different
promoter controls using the p-nitrophenol method. The enzyme activity produced by the FDH1 promoter was the highest, at
31.68 ± 0.24 U/mL, significantly higher than that obtained from the AOX1 (28.59 ± 0.37 U/mL) and FLD1 (25.88 ± 1.12 U/mL)
promoters (P ≤ 0.01). It is noteworthy that the activity levels driven by the AOX713 (25.37 ± 0.85 U/mL) and FLD1 promoters
were even lower than that of the commonly used AOX1 promoter (25.37 ± 0.85 U/mL). The activity of all the recombinant
strains was significantly higher than that of the negative control (pPIC9K empty vector, 0.96 ± 0.67 U/mL). These results
clearly indicated that the choice of promoter has a crucial impact on the production of lipase. Under the tested conditions,
the FDH1 promoter was proven to be the most effective choice for achieving high CALB activity (**Figure 17)**.

![](https://static.igem.wiki/teams/5790/engineer/17.webp)

**Figure 17** Lipase activity test result.

',
},
{
"title": 'Learn 2a',
"content": '

In this cycle, we systematically evaluated the performance of three promoters (AOX713, FDH1, FLD1) for the expression of
CALB in *P. pastoris* and enzyme activity. Therefore, this promoter will be included in the final round of
plasmid construction to maximize the yield of the recombinant CALB.

In this round of experiments, we realized that at the transcriptional level, the strength of the promoter is the main
determinant of the production of the recombinant protein, as it directly controls the amount of the synthesized mRNA [14].
Compared with the traditional AOX1 promoter and other tested candidate promoters, under the fermentation conditions adopted,
the FDH1 promoter is likely to exhibit stronger or more stable transcriptional activity, thereby promoting the accumulation
of higher levels of CALB mRNA and ultimately leading to an increase in protein synthesis and secretion.

',
},
]
},
{
"title": 'Cycle 2b: Signal peptide Optimization',
"content": '',
"children": [
{
"title": 'Design 2b',
"content": '

In the *P. pastoris* expression system, the signal peptide is crucial for guiding the secretion of heterologous
lipases to the extracellular space. It not only simplifies the downstream purification process but also significantly
increases the yield of the target protein [15]. In *P. pastoris*, the secretion of proteins by signal peptides begins
when the proteins are recognized by the signal recognition particles and the ribosome complexes are directed to the
endoplasmic reticulum. The newly synthesized peptide chains enter the endoplasmic reticulum lumen through the translocation
site, the signal peptide is removed, and the protein undergoes folding and processing in the Golgi apparatus before being
released into the extracellular space through exocytosis [16]. However, the compatibility of the signal peptides with
different exogenous proteins varies, and its cutting efficiency may differ depending on the N-terminal amino acid sequence
characteristics of the target protein, resulting in suboptimal secretion efficiency for some proteins or the presence of
additional amino acids at the N-terminus.

In this cycle, we obtained three hybrid signal peptides through literature research. These signal peptides were all modified
from the α-factor of *S. cerevisiae*, which is the most commonly used signal sequence in *P. pastoris* for
recombinant protein secretion. These signal precursor regions consist of two parts: the pro-peptide located at the
N-terminal of the precursor region, which functions as a signal peptide; and the precursor peptide (pre-peptide) located at
the C-terminal, which plays a role in promoting protein secretion (**Table 4)**.

**Table 4** Signal peptides used in this project.

|  |  |  |
| --- | --- | --- |
| Name | Source | Description |
| α-factor  (Control) | *S. cerevisiae* | It has a mature receptor-mediated secretion mechanism and is suitable for most exogenous proteins [17]. |
| 0030α-factor | Hybrid signal peptide | Preserves α-factor pro-sequence ER export signaling, 0030 pre-sequence boosts transmembrane translocation [17]. |
| SWP1-α-factor | Hybrid signal peptide | A hybrid signal peptide engineered based on the pro-sequence of the *P. pastoris* SWP1 protein\'s N-terminal 18 amino acids (MKLFFVGIVTTLLTLLVSC) fused to the α-factor signal peptide [18]. |
| KRE1-α-factor | Hybrid signal peptide | A hybrid signal peptide engineered from the N-terminal 18 amino acids of the *P. pastoris* cell wall synthesis-related protein KRE1 (MLNKLFIAILIVITAVIG) fused to the α-factor signal peptide pro-sequence [18]. |

To optimize the secretion efficiency of lipase in *P. pastoris*, this study replaced the original α-factor signal
peptide of the pPIC9K vector with engineered ones. We introduced three highly efficient secretion signal peptides that have
been verified in the literature: 0030α-factor, SWP1-α-factor, and KRE1-α-factor, which retained the AOX1 promoter on the
pPIC9K vector (**Figure 18**).

![](https://static.igem.wiki/teams/5790/engineer/18.webp)

**Figure 18** Plasmid Design diagram in cycle 2b.

',
},
{
"title": 'Build 2b',
"content": '

Firstly, we performed PCR amplification on each signal peptide, the *CALB* gene, and the plasmid backbone. The agarose gel
electrophoresis results demonstrated successful amplification of all target DNA fragments with the expected length: the
plasmid backbone fragments (Amp-0030α, Amp-SWP1α, Amp-KRE1α) are approximately 4.2 kb, the signal peptide-CALB fusion
fragments (0030α-CALB, SWP1α-CALB, KRE1α-CALB) are approximately 1.0 kb, and the Kanamycin resistance gene (Kan) is
approximately 4.7 kb (**Figure 19**).

**![](https://static.igem.wiki/teams/5790/engineer/19.webp)**

**Figure 19** The amplification results of the DNA fragments.

Following the amplification of the different signal peptides, *CALB* gene, and plasmid vector, we combined them
through homologous recombination. the transformation plates confirmed efficient plasmid introduction into
*E. coli* DH5α.

Colony PCR analysis demonstrated distinct bands of expected sizes: 0030-α-CALB (2426 bp), SWP1-α-CALB (2411 bp), and
KRE1-α-CALB (2411 bp). Sequencing results further verified the precise integration of each expression cassette with correct
promoter-signal peptide-CALB organization. These results collectively confirmed the accurate assembly of three plasmid
constructs, providing a reliable foundation for subsequent yeast transformation and protein expression studies (**Figure 20**).

![](https://static.igem.wiki/teams/5790/engineer/20.webp)

**Figure 20** The colony PCR and sequencing results of plasmids.

The recombinant plasmids were then successfully extracted and linearized by *SalI* enzyme before transformation, as
confirmed by agarose gel electrophoresis. Each plasmid displayed a single, clear band after single enzyme digestion,
corresponding to the expected size of the linearized vector. These linearized plasmids were then transferred into the
competent cells of *P. pastoris*. After 48 hours of cultivation, transformants of the yeast grew on the plates. We
conducted PCR on yeast colonies, the results showed the genomic integration, displaying specific bands of the expected size
(~954 bp) for all three signal peptide variants (0030α, SWP1α, KRE1α). These results collectively verified the accurate
construction of the engineered yeast strains (**Figure 21**).

**![](https://static.igem.wiki/teams/5790/engineer/21.webp)**

**Figure 21** Construction result of *P. pastoris* containing lipase.

(A) The linearization results of the plasmids. (B) Transformation plates. (C) Yeast colony PCR results.

',
},
{
"title": 'Test 2b',
"content": '

* **Lipase secretion and quantitative analysis**

Next, we conducted fermentation on each of these three strains. The SDS-PAGE results showed distinct protein bands at
approximately 43.8 kDa in the 72-hour samples for all engineered strains, corresponding to secreted CALB, while absent in
the negative control (pPIC9K) and pre-induction samples. Quantitative analysis revealed that the 0030-α signal peptide
yielded the highest CALB concentration (90.76 ± 3.65 μg/mL), significantly outperforming the standard α-factor signal
peptide (68.21 ± 4.94 μg/mL, ​​P ≤ 0.001). The SWP1-α variant also showed improved secretion (69.62 ± 5.13 μg/mL), whereas
KRE1-α resulted in lower yield (56.28 ± 1.79 μg/mL). These results demonstrated that signal peptide engineering
significantly enhances CALB secretion efficiency in *P. pastoris*.

**![](https://static.igem.wiki/teams/5790/engineer/eng-22.webp)**

**Figure 22** (A)SDS-PAGE verification and (B) quantification of CALB under different promoter regulation.

* **Enzyme activity test of lipase**

The enzyme activity test results showed that the enzyme activity of 0030-α-CALB was the highest (31.25 U/mL), which was
significantly higher than that of the standard α-factor control group (28.59 U/mL), with a statistically significant
difference (\*P ≤ 0.05). The activities of SWP1-α-CALB (27.32 U/mL) and KRE1-α-CALB (27.08 U/mL) variants were comparable to
that of the α-factor control group, and no significant differences were observed between them (ns). These results indicate
that the 0030-α signal peptide enhances the catalytic efficiency of CALB (**Figure 23)**.

**![](https://static.igem.wiki/teams/5790/engineer/23.webp)**

**Figure 23** Lipase activity test result.

',
},
{
"title": 'Learn 2b',
"content": '

This study systematically evaluated the efficiency of three engineered signal peptides (0030α-factor, SWP1-α-factor, and
KRE1-α-factor) in directing CALB secretion in *P. pastoris*. Recombinant plasmids containing different signal
peptides were successfully constructed and transformed into *P. pastoris*. SDS-PAGE and quantitative analysis
revealed that the 0030α-signaling peptide induced the highest CALB secretion concentration (90.76 ± 3.65 μg/mL),
significantly outperforming the traditional α-factor signaling peptide. Enzyme activity assays further confirmed that
0030α-CALB exhibited markedly higher activity (31.25 U/mL) compared to the control group. Therefore, 0030α-signal peptide
was identified as the optimal signal peptide and will be used for final recombinant strain construction.

',
},
]
},
{
"title": 'Cycle 3: Building and Testing the Optimal Combination',
"content": '',
"children": [
{
"title": 'Design 3',
"content": '

The best-performing combinations were finally integrated into expression cassettes for recombinant strain construction. As
shown in **Figure 24,** the expression vector pPIC9K was engineered to contain the optimal FDH1 promoter and
0030α-factor signal peptide fused with the *CALB* gene.

![](https://static.igem.wiki/teams/5790/engineer/24.webp)

**Figure 24** Plasmid Design diagram in cycle 3.

',
},
{
"title": 'Build 3',
"content": '

The agarose gel electrophoresis results (**Figure 25A**) confirmed successful amplification of both the vector
backbone (Amp-FDH1, ~4402 bp) and the insert fragment (0030α-CALB-Kan, ~5954 bp), with bands matching expected sizes. Colony
PCR verification demonstrated successful transformation of *E. coli* DH5α competent cell, showing specific bands of
approximately 2495 bp corresponding to the target expression cassette in multiple transformants (**Figure 25B-C**). The sequencing results validated the accurate assembly of the optimized expression system combining the FDH1 promoter
and 0030α-factor signal peptide (**Figure 25D**).

![](https://static.igem.wiki/teams/5790/engineer/25.webp)

**Figure 25** The amplification results of the DNA fragments.

(A) Agarose gel electrophoresis result. (B-D) Colony PCR and sequencing results.

We then extracted and linearized the correct plasmids for yeast transformation. The agarose gel electrophoresis (**Figure 26**
**A**) confirmed successful linearization, showing a single clear band for the linearized plasmid (lane 2)
compared to the negative control (lane 1). The transformation plates (**Figure 26** **B**)
exhibited robust colony growth, indicating efficient plasmid introduction into *P. pastoris*. Furthermore, yeast
colony PCR analysis (**Figure 26** **C**) validated the genomic integration, revealing a distinct
band at approximately 954 bp corresponding to the FDH1-0030α-CALB expression cassette. These results collectively confirm
the successful construction of the recombinant *P. pastoris* strain.

![](https://static.igem.wiki/teams/5790/engineer/26.webp)

**Figure 26** Construction result of *P. pastoris* containing lipase**.**

(A) Plasmid Linearization result. (B) Transformation plates. (C) Yeast colony PCR result.

',
},
{
"title": 'Test 3',
"content": '

* **Lipase secretion and quantitative analysis**

Next, we conducted fermentation on each of these strains (FDH1-0030α-CALB belongs to the experimental group, while
FDH1-α-CALB and AOX1-0030α-CALB constitute the control group) and analyzed CALB secretion. The SDS-PAGE results (**Figure 27A**) clearly showed distinct protein bands at approximately 43.8 kDa in the experimental groups, corresponding to the expected
molecular weight of CALB, while absent in the negative control. Quantitative analysis (**Figure 27B**)
demonstrated that the combination of the FDH1 promoter and 0030-α-factor signal peptide yielded the highest secretion level
(107.02 ± 3.95 μg/mL), significantly outperforming the other constructs (​​P ≤ 0.01). These results confirmed the synergistic
effect of promoter and signal peptide optimization on CALB production.

![](https://static.igem.wiki/teams/5790/engineer/27.webp)

**Figure 27** SDS-PAGE verification of CALB secretion.

(A) SDS-PAGE result. (B) Quantitative results.

* **Enzyme activity test of lipase**

Similarly, we conducted an enzyme activity test. The results showed that compared with the negative control pPIC9K (NC), the
lipase activity of all the recombinant strains was significantly increased. Among them, the combination of the FDH1 promoter
and the 0030-α-factor signal peptide (FDH1-0030-α-factor-CALB) exhibited the highest enzyme activity (33.41 U/mL).
Statistical analysis indicated that the enzyme activity of this combination was significantly higher than that of the
strains that only optimized the promoter (FDH1-α-factor-CALB, 31.68 U/mL) or only optimized the signal peptide
(AOX1-0030-α-factor-CALB, 31.25 U/mL) (P ≤ 0.05), confirming the positive effect of the synergistic optimization of the
promoter and signal peptide on enhancing the catalytic efficiency of the lipase (**Figure 28**).

![](https://static.igem.wiki/teams/5790/engineer/28.webp)

**Figure 28** Lipase activity test results.

Finally, we investigated the temperature and pH values of the reaction to determine the optimal catalytic conditions
of CALB. As shown in **Figure 29A,** the temperature optimization experiment indicated that CALB exhibited the highest
enzymatic activity (approximately 45 U/mL) at 45°C, while the activity significantly decreased at low temperatures
(25°C) or high temperatures (≥65°C). As shown in **Figure 29B,** the pH optimization results revealed that the enzyme
maintained high activity within the range of 7.5–9.0, with the optimal pH being 8.0. It is noteworthy that CALB
could still maintain over 70% relative activity within a wide pH range (6.0–10.0), indicating its good
acid-base stability. These data clearly defined the optimal reaction conditions of CALB as 45°C and a neutral
to weakly alkaline environment, providing key parameters for its industrial application.

![](https://static.igem.wiki/teams/5790/engineer/20-new.webp)

**Figure 29** Optimization of (A) reaction temperature and (B) pH for CALB.

',
},
{
"title": 'Learn 3',
"content": '

In this project, we successfully established an efficient system for recombinant CALB production in *P.
pastoris.* The optimized gene construct, which combines the **FDH1 promoter** and the **0030α factor signal
peptide** of the *CALB* gene, was successfully integrated into the yeast genome. The secretion level of CALB
reached 107.02 ± 3.95 μg/mL, and the enzyme activity was 33.41 U/mL, which were **1.84 times** and **1.17 times**
higher than those of the original strain (AOX1-α factor-CALB, secretion: 68.21 μg/mL, enzyme activity: 28.59 U/mL),
respectively. In addition, we determined that **the optimal catalytic conditions for CALB were 45°C and a
pH value of 8.0.** Under these conditions, the enzyme activity could reach up to 44.96 U/mL, and
the enzyme showed high stability within a wide pH range (6.0-10.0). In conclusion, the collaborative
optimization of the FDH1 promoter and the 0030α factor signal peptide is the key strategy for achieving
high-yield active CALB in *P. pastoris.* This recombinant strain and the clearly defined optimal reaction
parameters have laid a solid foundation for the industrial application of this biocatalyst.

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

[1] Zhang, N., Suen, W. C., Windsor, W., Xiao, L., Madison, V., & Zaks, A. (2003). Improving tolerance of Candida
antarctica lipase B towards irreversible thermal inactivation through directed evolution. Protein Engineering, 16(8),
599-605.

[2] Yan, J., Yang, J., Xu, L., & Yan, Y. (2007). Gene cloning, overexpression and characterization of a novel organic
solvent tolerant and thermostable lipase from Galactomyces geotrichum Y05. Journal of Molecular Catalysis B: Enzymatic,
49(1), 28-35.

[3] Zheng, J., Liu, C., Liu, L., & Jin, Q. (2013). Characterisation of a thermo-alkali-stable lipase from
oil-contaminated soil using a metagenomic approach. Systematic and Applied Microbiology, 36(3), 197-204.

[4] Gu L, Zhang J, Liu B, et al. High-Level extracellular production of glucose oxidase by recombinant *Pichia pastoris* using a combined strategy[J]. Applied Biochemistry and Biotechnology, 2015, 175(3): 1429-1447.

[5] Qiang L, Wang H, Farmer SR. Adiponectin secretion is regulated by SIRT1 and the endoplasmic reticulum oxidoreductase
Ero1-Lα[J]. Molecular and Cellular Biology, 2007, 27(13): 4698-4707.

[6] Onda Y, Kobori Y. Differential activity of rice protein disulfide isomerase family members for disulfide bond formation
and reduction[J]. FEBS Open Bio, 2014, 4(1): 730-734.

[7] Wang X, Wang P, Li W, Zhu C, Fan D. Effect and mechanism of signal peptide and maltose on recombinant type III collagen
production in Pichia pastoris. Appl Microbiol Biotechnol. 2023 Jul;107(13):4369-4380.

[8] EasySelect Pichia Expression Kit,Rev.Date 18 June 2010, Manual part no.25-0172.

[9] Vogl T, Hartner FS, Glieder A. New opportunities by synthetic biology for biopharmaceutical production in Pichia
pastoris. Curr Opin Biotechnol. 2013 Dec;24(6):1094-101.

[10] Vogl, T., & Glieder, A. (2013). Regulation of Pichia pastoris promoters and its consequences for protein
production. New Biotechnology, 30(4), 385-404.

[11] Liu, Q., Li, Y.-h., Tao, L.-f., Yang, J.-y., Zhang, Y.-l., & Cai, M.-h. (2024). Rational design and
characterization of enhanced alcohol-inducible synthetic promoters in Pichia pastoris. Applied and Environmental
Microbiology, 91(1), e02191-02124. doi:10.1128/aem.02191-24

[12] HU Liyuan, LIU Luyao, BAI Zhonghu,et al.Cofactor specificity engineering and application of formate dehydrogenase from
Komagataella phaffii[J].Food and Fermentation Industries,2023,49(13)：9-16.

[13] CREGG JAMES M. Promoter for the Pichia pastoris formaldehyde dehydrogenase gene FLD1. United States Reexamination
Certificate First Reexamination US19990345828. 02 Jul 1999.

[14] Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol
Rev. 2000 Jan;24(1):45-66.

[15] Dai H, Zhang C, Wu J, Tang Q, Xie Y, Yu Y, Lin Y, Huang Y. Optimizing Pichia pastoris protein secretion: Role of
N-linked glycosylation on the α-mating factor secretion signal leader. J Biotechnol. 2024 Aug 10;391:1-10.

[16]
<https://www.tutorialspoint.com/signal-peptides-an-overview>

[17] Lv, X., Zhang, Y., Wang, L., Cui, S., Liu, Y., Li, J., Liu, L. (2024). Expression and antimicrobial activity of the
recombinant bovine lactoferricin in Pichia pastoris. Synthetic and Systems Biotechnology, 9(1), 26-32.

[18] Richard Zahrl, Wien (AT); Oezge Ata Aykol, Wien (AT); Diethard Mattanovich,Wien (AT); Brigitte Gasser, Wien (AT)signal
peptides for increased protein secretion\_patent.US20240141363A1. 02 May 2024.

',
"children": []
},
] %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}
![](https://static.igem.wiki/teams/5790/common/engineering.webp)
{% endblock %}
{% block page\_content %}
{% endblock %}

{% extends "layout.html" %}
{% set info\_data = [
{
"title": '1.Microbial Media',
"content": '

## 1.1 LB Luquid Media

|  |  |
| --- | --- |
| **Component** | **Concentration** |
| Peptone | 10 g/L |
| Yeast Extract | 5 g/L |
| NaCl | 10 g/L |

Solid media supplemented with 15–20 g/L agar

## 1.2 YPD Luquid Media

|  |  |
| --- | --- |
| **Component** | **Concentration** |
| Peptone | 20 g/L |
| Yeast Extract | 10 g/L |
| Glucose | 20 g/L |

Solid media supplemented with 15–20 g/L agar

## 1.3 BMGY Luquid Media

|  |  |
| --- | --- |
| **Component** | **Concentration** |
| Peptone | 20 g/L |
| Yeast Extract | 10 g/L |
| YNB | 6.7 g/L |
| Biotin | 0.4 mg/L |
| Glycerol | 10 g/L |
| Phosphate-buffered solution | 100 mmol/L |

## 1.4 BMMY Luquid Media

|  |  |
| --- | --- |
| **Component** | **Concentration** |
| Peptone | 20 g/L |
| Yeast Extract | 10 g/L |
| YNB | 6.7 g/L |
| Methanol | 100 mL/L |
| Biotin | 0.4 mg/L |
| Phosphate-buffered solution | 100 mmol/L |

## 1.5 MD Culture media

|  |  |
| --- | --- |
| **Component** | **Concentration** |
| YNB | 13.4 g/L |
| D-biotin | 0.4 mg/L |
| D-glucose | 20 g/L |

',
"children": []
},
{
"title": '2. PCR Amplification',
"content": '

## 2.1 PCR System and Reaction Procedure:

|  |  |  |
| --- | --- | --- |
| **Reagent** | **Volume (μL)** | **Procedure** |
| PrimeStar Max Premix (2×) | 25 | Pre-denaturation: 98 ℃ for 5 min |
| Primer F (10 μM) | 1 | Denaturation: 98 ℃ for 10 sec |
| Primer R (10 μM) | 1 | Annealing: Tm for 15 sec |
| DNA template | 1 (10 ng) | Extension: 72 ℃, 30 sec/kb |
| ddH2O | 22 | Back to step 2 (30 times) |
| Total | up to 50 | Final elongation: 72 ℃ for 2 min |
|  |  | 10 ℃ hold |

## 2.2 Primer

(1) lipases:

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| **Amplified fragment** | **Template** | **Length**  **(bp)** | **Primer** | **Tm (℃)** | **Final**  **Tm (℃)** | **Time**  **（min）** |
| CALB | P-CALB | 954 | CALB-SEQ-F | 57 | 58 | 1 |
| CALB-SEQ-R | 62 |
| GGL | P-GGL | 1692 | GGL-SEQ-F | 55 | 57 | 1.5 |
| GGL-SEQ-R | 57 |
| LipZ01 | P-LipZ01 | 1428 | LZ-SEQ-F | 55 | 54 | 1.5 |
| LZ-SEQ-R | 53 |
| pPICZαA-vector | pPICZαA | 3476 | ZαA-F | 59 | 59 | 2 |

(2) Promoters：

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| **Amplified fragment** | **Template** | **Length**  **(bp)** | **Primer** | **Tm (℃)** | **Final**  **Tm (℃)** | **Time**  **（min）** |
| AOX713 | pPIC9K-AOX713 | 4366 | 9K-Amp-F | 56 | 57 | 3 |
| Amp-Promotor-R | 69 |
| FDH1 | pPIC9K-FDH1 | 4661 | 9K-Amp-F | 56 | 57 | 3 |
| Amp-Promotor-R | 69 |
| FLD1 | pPIC9K-FLD1 | 4244 | 9K-Amp-F | 56 | 57 | 3 |
| Amp-Promotor-R | 69 |
| CALB | 2\_CALB\_pPICZαA | 996 | CALB-SEQ-F | 57 | 58 | 1 |
| CALB-His-9k-R1 | 65 |
| Kan | pPIC9K-AOX713 | 4699 | 9K-Kan-F | 59 | 57 | 3 |
| 9K-Kan-R | 56 |

(3) Signal peptides：

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| **Amplified fragment** | **Template** | **Length**  **(bp)** | **Primer** | **Tm (℃)** | **Final**  **Tm (℃)** | **Time**  **（min）** |
| 0030 | pPIC9K-AOX1-0030 | 4197 | 9K-Amp-F | 56 | 55 | 3 |
| Amp-0030-R | 54 |
| 0030-CALB | 2\_CALB\_pPICZαA | 1015 | 0030-CALB-F | 63 | 64 | 1 |
| CALB-His-9k-R | 65 |
| SP4 | pPIC9K-AOX1-SP4 | 4182 | 9K-Amp-F | 56 | 57 | 3 |
| Amp-SP4-14-R | 60 |
| SP14 | pPIC9K-AOX1-SP14 | 4182 | 9K-Amp-F | 56 | 57 | 3 |
| Amp-SP4-14-R | 60 |
| SP4/14-CALB | 2\_CALB\_pPICZαA | 1020 | SP4-14-CALB-F | 53 | 54 | 1 |
| 9K-Kan-R | 56 |

',
"children": []
},
{
"title": '3. Homologous Recombination',
"content": '

## 3.1 Calculation of Homologous Recombination System Usage

Amount of Linearized Vector and Insert Fragment

(1) Entry cloning and single-fragment homologous recombination reaction:

Optimal amount of cloning vector = [0.02 × cloning vector base pairs] ng (0.03 pmol)

Optimal amount of insert fragment = [0.04 × insert fragment base pairs] ng (0.06 pmol)

(2) Multi-fragment homologous recombination reaction (2–5 fragments):

Optimal amount of cloning vector = [0.02 × cloning vector base pairs] ng (0.03 pmol)

Optimal amount of each fragment = [0.02 × each fragment base pairs] ng (0.03 pmol)

## 3.2 Preparation of Homologous Recombination System

|  |  |
| --- | --- |
| **Component** | **Reaction** |
| Linearized plasmid | X μl |
| Inserted fragments (n≤5) | Y1+Y2+…+Yn μl |
| 2×ClonExpress Mix | 5 μl |
| ddH2O | to 10 μl |

Prepare the mixture according to the above system based on the actual amount required, then incubate at 50°C for 30 minutes.

',
"children": []
},
{
"title": '4. Agarose Gel Electrophoresis and DNA Gel Extraction',
"content": '

## 4.1 Agarose gel electrophoresis

(1) Weigh agarose powder and add it to electrophoresis buffer (TAE). Heat in a microwave until the solution becomes
completely transparent.

(2) Add nucleic acid stain (YeaGreen).

(3) Pour the gel solution into the casting tray, insert a comb, and allow it to solidify at room temperature for 15 minutes.

(4) After solidification, remove the comb and place the gel into the electrophoresis tank. Add buffer until the gel is
submerged by 1–2 mm.

(5) Load the PCR-amplified DNA samples into the wells, with DNA marker added to the first well.

(6) Run the electrophoresis at 180 V for 15 minutes, ensuring correct electrode orientation.

(7) After electrophoresis, place the gel under a blue-light gel cutter to observe the bands.

## 4.2 DNA Gel Extraction

4.2.1 Materials: Binding column, collection tube, Buffer B2, wash solution, elution buffer

4.2.2 Procedure:

(1) Excise the gel slice containing the target DNA band and weigh it.

(2) Add 3–6 volumes of Buffer B2 (relative to the gel weight) and incubate at 50 °C for 5–10 minutes to dissolve the gel.

(3) For fragments <500 bp, add isopropanol equivalent to 1/3 volume of Buffer B2.

(4) Transfer the dissolved gel solution into the binding column and centrifuge at 8,000 × g for 30 seconds. Discard the
flow-through.

(5) Add 500 μl of wash solution and centrifuge at 9,000×g for 30 seconds. Discard the flow-through.Repeat Step 5 once.

(6) Centrifuge the empty column at 9,000×g for 1 minute to dry.

(7) Transfer the column to a clean 1.5 ml centrifuge tube. Apply 15–40 μl of elution buffer to the center of the column
membrane. Incubate at room temperature for 1 minute, then centrifuge for 1 minute. Collect and store the eluted DNA
solution.

',
"children": []
},
{
"title": '5. *E. coli* Transformation ',
"content": '

## 5.1 One-Step Preparation of Competent *E. coli* Cells

Pre-treatment: Mix 2× BT Buffer T and sterile distilled water in equal volume and pre-chill on ice. Use 100 μl of the
diluted 1× BT Buffer T per 1 ml of bacterial culture.

(1) Pick a single colony from a plate or use frozen glycerol stock to inoculate 2–5 ml of appropriate medium. Incubate
overnight at 37°C.

(2) Dilute the overnight culture 1:100 into pre-warmed fresh LB medium. Shake at 250 rpm and 37°C until early log phase
(OD600 = 0.3–0.4) is reached, typically 1.5–3 hours.

(3) Centrifuge the culture at 3,500–5,000 rpm and 4°C for 5–10 minutes to harvest the cells.

(4) Resuspend the pellet thoroughly in the pre-chilled 1× BT Buffer T (100 μl per ml of original culture). Incubate on ice
for 10 minutes. The competent cells can be used directly for transformation or aliquoted, flash-frozen in liquid nitrogen,
and stored at -70°C.

## 5.2 Plasmid Transformation

(1) Add 10 μl of the recombinant product to 100 μl of competent cells. Mix gently.

(2) Incubate on ice for 30 minutes to allow DNA adsorption.

(3) Apply heat shock at 42°C for 90 seconds to facilitate DNA uptake.

(4) Immediately return the tube to ice for 2 minutes.

(5) Add 900 μl of LB broth (without antibiotic) and incubate at 37°C with shaking for 45–60 minutes for recovery.

(6) Spread appropriate volumes onto selective plates under sterile conditions

',
"children": []
},
{
"title": '6. Plasmid DNA Extraction',
"content": '

## 6.1 Materials

Binding column, collection tube, Buffer S, Buffer SP1, Buffer SP2, Buffer SP3, Buffer DW1, Wash Solution, Elution Buffer,
RNase A

## 6.2 Procedure

(1) Preparation:

Ensure RNase A has been added to Buffer SP1.

Confirm that absolute ethanol has been added to the Wash Solution.

Check for precipitation in Buffers SP2 and SP3; redissolve if necessary.

(2) Column equilibration: Add 500 µl of Buffer S to the binding column (placed in a collection tube). Centrifuge at 12,000 ×
g for 1 minute. Discard the flow-through and reassemble the column with the same collection tube.

(3) Harvest 1.5–5 ml of overnight bacterial culture by centrifugation at 8,000 × g for 2 minutes. Discard the supernatant
completely.

(4) Resuspend the pellet thoroughly in 250 µl of Buffer SP1.

(5) Add 250 µl of Buffer SP2 and immediately mix by inverting the tube gently 5–10 times. Incubate at room temperature for
2–4 minutes.

(6) Add 350 µl of Buffer SP3 and mix immediately by inverting the tube gently 5–10 times.

(7) Centrifuge at 12,000 × g for 5–10 minutes. Transfer the supernatant to the equilibrated binding column and centrifuge at
8,000 × g for 30 seconds. Discard the flow-through.

(8) Add 500 µl of Buffer DW1 and centrifuge at 9,000 × g for 30 seconds. Discard the flow-through.

(9) Add 500 µl of Wash Solution and centrifuge at 9,000 × g for 30 seconds. Discard the flow-through.

(10) Repeat Step 9 once.

(11) Centrifuge the empty column at 9,000 × g for 1 minute to dry.

(12) Transfer the column to a clean 1.5 ml microcentrifuge tube. Apply 50–100 µl of Elution Buffer to the center of the
membrane. Incubate at room temperature for 1 minute, then centrifuge at 9,000 × g for 1 minute. Collect and store the eluted
DNA solution

',
"children": []
},
{
"title": '7. Vector Linearization',
"content": '

(1) Set up the reaction according to the following system and incubate at 37 °C for 1 hour.

|  |  |
| --- | --- |
| **Reagent** | **Volume (μL)** |
| 10×FuniCut™ Color Buffer | 4 |
| Plasmid | 20 |
| SalI | 2 |
| ddH2O | 14 |
| Total | 40 |

(2) Verify linearization by agarose gel electrophoresis.

(3) Purify the linearized vector using a gel extraction kit.

',
"children": []
},
{
"title": '8. *Pichia pastoris* Transformation',
"content": '

## 8.1 Preparation of Yeast Competent Cells

(1) Strain activation: Streak glycerol stock of *P. pastoris* stored at -80 °C onto a YPD agar plate. Incubate
inverted at 30 °C for 2–4 days to activate the strain.

(2) Inoculation: Pick a single fresh yeast colony using a sterile loop or tip and inoculate into 10 ml of YPD liquid medium.

(3) Culture: Incubate at 28–30 °C with shaking at 250 rpm overnight.

(4) Subculture: Transfer the overnight culture into 50 ml of fresh YPD liquid medium to an initial OD600 of
0.1–0.2. Culture at 30 °C, 250–300 rpm for 4–6 hours until OD600 reaches 0.6–0.8 (mid-log phase).

(5) When OD600 reaches 0.6–0.8, centrifuge the culture at 3,000 × g for 3 minutes at room temperature. Discard
supernatant and retain pellet.

(6) Resuspend the pellet in 5 ml of Buffer A. Centrifuge at 3,000 × g for 3 minutes at room temperature. Discard
supernatant.

(7) Resuspend the pellet in 400 μl of Buffer A and aliquot 100 μl into sterile 1.5 ml microcentrifuge tubes.

(8) Add 5.5 μl of DMSO to each tube and mix gently. The competent cells can be used directly for transformation or slowly
frozen using a programmed freezing container at -80 °C for long-term storage.

## 8.2 *Pichia pastoris* Transformation — LiCl Method

(1) Mix 3 μg of linearized plasmid DNA (in 10 μl) with 5 μl of heat-denatured Carrier DNA by pipetting. Add the mixture to
50 μl of competent cells. The total volume of DNA added should not exceed 10 μl.

(2) Incubate in a 37 °C water bath for 5 minutes. Invert tube gently 1–2 times during incubation until frozen cells are
fully thawed.

(3) Remove from water bath and add 750 μl of Buffer B. Mix gently by inverting the tube.

d. Incubate in a 30 °C water bath for 1 hour. Gently mix by inverting or flicking the tube every 15 minutes to improve
transformation efficiency.

(4) Centrifuge at 2,000 × g for 10 minutes at room temperature. Discard supernatant and retain pellet. Resuspend the pellet
in 750 μl of Buffer C.

(5) Centrifuge at 2,000 × g for 10 minutes. Discard supernatant and resuspend the pellet gently in 200 μl of Buffer C.

(6) Spread the entire resuspension evenly onto solid selective medium with appropriate antibiotics. Incubate inverted at
30 °C for 3–4 days until single colonies appear.

',
"children": []
},
{
"title": '9. Colony PCR Verification',
"content": '

Pick single colonies from the transformation plate and prepare the reaction mixture according to the following system. Run
the corresponding PCR program.

|  |  |  |
| --- | --- | --- |
| **Reagent** | **Volume (μL)** | **Procedure** |
| 2×Hieff® Ultra-Rapid II HotStart PCR Master Mix | 5 | Pre-denaturation: 95℃for 10 min |
| Forward Primer (10 μM) | 0.4 | Denaturation: 95 ℃ for 5 sec |
| Reverse Primer (10 μM) | 0.4 | Annealing: 57℃ for 15 sec |
| ddH2O | 4.2 | Extension: 72 ℃, 3 sec/kb |
| Template | Single colony | Back to step 2 (20 times) |
| Total | 10 | Final elongation: 72 ℃ for 2 min |
|  |  | 10 ℃ hold |

',
"children": []
},
{
"title": '10. Protein Secretion and SDS-PAGE',
"content": '

## 10.1 Yeast Culture

(1) Inoculate the strain into YPD liquid medium and incubate overnight. Then transfer 1% of the culture into 3 mL of BMGY
medium and incubate overnight until OD600 reaches approximately 10.

(2) Harvest the cells by centrifugation at 4,000 rpm for 5 minutes. Resuspend the pellet in 5 mL of BMMY induction medium
and retain 100 µL as the initial sample.

(3) Culture at 30 °C with shaking at 250 rpm. Take 100 µL samples every 24 hours and supplement with methanol to a final
concentration of 1% each time after sampling.

## 10.2 SDS-PAGE for Protein Expression Analysis

(1) Assemble the gel-casting apparatus. Begin by preparing the lower gel (for a 1.50 mm mini-gel).

(2) Mix 4 mL of lower gel solution (2X) with 4 mL of lower gel buffer (2X) in a gel preparation cup.

(3) Add 80 µL of modified polymerization accelerator to the mixture from step 2. Stir gently to avoid bubbles.

(4) Pour the mixed lower gel solution into the gel mold until the liquid is 1.5 cm below the top of the glass plates.
Carefully overlay with water or alcohol to flatten the surface and minimize exposure to air.

(5) Let it stand at room temperature (25 °C) for 6–10 minutes. A visible interface between the gel and overlay indicates
polymerization.

(6) Prepare the upper gel (for a 1.50 mm mini-gel).

(7) Slowly remove the overlay solution. Mix 1.0 mL of upper gel solution (2X) with 1.0 mL of colored upper gel buffer (2X)
in a new cup.

(8) Add 20 µL of modified polymerization accelerator to the mixture from step 7. Mix gently without introducing bubbles.

(9) Pour the upper gel solution onto the polymerized lower gel until full. Slowly insert the comb, avoiding bubbles.

(10) Allow the upper gel to polymerize for 10–15 minutes. Carefully remove the comb and rinse the wells with electrophoresis
buffer using a pipette tip or syringe. Proceed with SDS-PAGE electrophoresis.

## 10.3 Protein Sample Treatment

(1) Preparation of Fermentation Supernatant: Following the fermentation process, the culture broth was centrifuged at 12,000 rpm
for 5 min to remove yeast cells and large particulate matter. The resulting supernatant, containing the secreted recombinant protein,
was carefully collected for further analysis.

(2) Sample Denaturation and Reduction: 16 μL supernatant was mixed with 4 μL 5×protein loading buffer, which typically contains SDS,
glycerol, bromophenol blue, and a reducing agent like β-mercaptohenol . The mixture was heated at 95°C for 10 min to denature the protein.

(3) Sample loading and electrophoresis: Take 10 μl of the treated protein sample and add it to the wells of the protein gel.
Electrophoresis was initiated at a constant current of 80 V through the stacking gel, then increased to 120 V through the
separating gel until the dye front reached the bottom.

(4) Staining and Visualization After electrophoresis, the gel was carefully removed from the plates and immersed in a Coomassie
Brilliant Blue Rapid Staining Solution for 10 min to fix and stain the proteins. The gel was then destained using water until
the protein bands were clear against a transparent background.

',
"children": []
},
{
"title": '11. Lipase Purification',
"content": '

(1) Label the protein sample before binding to the resin as "S".

(2) Incubate the protein sample with Ni-NTA resin on ice with gentle shaking for 1 hour.

(3) After binding, centrifuge the mixture at 1,000 × g for 10 seconds. Transfer the supernatant to a new tube and label as
"FT".

(4) Add 50 µl of Wash Buffer to the resin, centrifuge at 1,000 × g for 10 seconds, and transfer the supernatant to a new
tube labeled "W".

(5) Repeat Step 4 and combine the supernatant with the previous wash fraction.

(6) Add 30 µl of Elution Buffer to the resin, centrifuge at 1,000 × g for 10 seconds, and transfer the supernatant to a new
tube labeled "E".

(7) Repeat Step 6 and combine the eluate with the previous elution fraction.

(8) Prepare samples for SDS-PAGE by mixing 20 µl of each sample (S, FT, W, E) with 5 µl of 5× SDS-PAGE Loading Buffer.

(9) Denature the samples in a PCR instrument at 98°C for 5 minutes. Store temporarily at -20°C.

',
"children": []
},
{
"title": '12. BCA assay',
"content": '

## 12.1 Reagent Preparation

(1) Calculate the total volume of BCA working solution required using the following formula:

Total volume of BCA working solution = (Number of standard points + Number of samples) × Number of replicates × Volume of
BCA working solution required per sample.

(2) Based on the calculated total volume, pipette Solution A and Solution B at a ratio of 50:1. Mix well to prepare the BCA
working solution.

(3) Dilute a certain amount of the BSA standard solution to a concentration of 500 μg/ml using 1X PBS buffer.

(4) Label 16 microcentrifuge tubes, setting up for 8 duplicate pairs. Process the tubes in parallel according to the table
below. Dilute the samples with 1X PBS to an appropriate concentration for the assay.

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Number**  **Solution** | **0** | **1** | **2** | **3** | **4** | **5** | **6** | **7** |
| 1×PBS (μL) | 300 | 285 | 270 | 240 | 180 | 120 | 60 | 0 |
| 0.5 mg/mL BSA (μL) | 0 | 15 | 30 | 60 | 120 | 180 | 240 | 300 |
| Concentration of BSA (μg/mL) | 0 | 25 | 50 | 100 | 200 | 300 | 400 | 500 |

12.2 Protein Concentration Assay

(1) Select 20 wells on the microplate, divided into standard and sample groups. For the standard group (16 wells), add 20 μl
of the corresponding concentration of standard protein solution to each well, with one replicate per concentration. For the
remaining 4 wells (sample group), assign them into 2 duplicate pairs (wells with the same ID are replicates). Add 20 μl of
differently diluted sample solutions to the wells with different IDs.

(2) Add 200 μl of the BCA working solution to each well and mix thoroughly immediately.

(3) Incubate the plate at 37°C in a water bath for 30 minutes.

(4) After cooling to room temperature, measure the absorbance (A562) of each well using a microplate reader.

(5) Generate a standard curve by plotting the average A562 value for each standard concentration (y-axis) against
the corresponding protein concentration (x-axis). Calculate the protein concentration of the diluted samples from the
standard curve using the average A562 value of the duplicate measurements. Select the dilution factor that yields a reading
within the linear range of the standard curve. Multiply the calculated concentration by the dilution factor to determine the
final protein concentration of the original sample.

',
"children": []
},
{
"title": '13. Lipase Activity Assay',
"content": '

## 13.1 Standard Curve for Enzyme Activity

(1) p-Nitrophenol (p-NP) Stock Solution (2 mmol/L)

20 mmol/L: Weigh 0.014 g p-nitrophenol (p-NP) and dissolve in 5 mL PB buffer (pH 8.0, containing 1% Triton X-100). Store in
a brown bottle at 4°C.

2 mmol/L: Dilute the 20 mmol/L stock solution 10-fold using the same PB buffer. Store in a brown bottle at 4°C.

(2) Standard Curve Preparation (in triplicate)

Take 0.01, 0.02, 0.03, 0.04, 0.06, and 0.08 mL of the 2 mmol/L p-NP stock solution and dilute each to 2 mL with PB buffer
(pH 8.0, 1% Triton X-100). Measure the absorbance at 410 nm.

Plot a standard curve with p-NP concentration (0.01, 0.02, 0.03, 0.04, 0.06, 0.08 mmol/L) on the x-axis and absorbance (y)
on the y-axis.

## 13.2 Lipase Activity Measurement

(1) Solution Preparation

Solution A (3 mg/mL): Dissolve 30.0 mg p-nitrophenol palmitate (p-NPP) in 10.00 mL isopropanol. Store at 4°C in a brown
bottle or a foil-wrapped 15 mL tube.

Solution B: Add 1 drop of Triton X-100 to 100 mL PB buffer (pH 8.0). Mix well and store at 4°C.

(2) Activity Assay (in triplicate, using a microplate reader)

Mix 1 mL Solution A with 9 mL PB buffer (pH 8.0, 1% Triton X-100) in a tube. Incubate at 37 °C for 5 minutes. Add 190 µL
Reaction Mix to each well of a 96-well plate. Then add:10 µL sample, 10 µL PB buffer, 10 µL heat-inactivated enzyme (boiled
for 5 min). Incubate at 37 °C for 10 minutes and measure absorbance at 410 nm. Perform each measurement in triplicate and
calculate the average.

(3) Definition of Lipase Activity Unit (U):

One unit (U) of lipase activity is defined as the amount of enzyme required to release 1 μmol of p-nitrophenol (p-NP) per
minute from p-nitrophenol palmitate (p-NPP) under assay conditions (410 nm). The calculation formula is:

Activity (U/mL)

Where:

*y*: Absorbance value of the sample

*b*: Absorbance of the blank control

*k*: Slope of the standard curve (from p-NP standard curve)

*n*: Dilution factor (n = 1 if undiluted)

*V*: Total reaction volume (in mL). \*Note: Here the total reaction volume in the well is 0.2 mL (190 µL Reaction Mix
+ 10 µL sample)\*

1000: Conversion factor from mmol to μmol

*t*: Reaction time (in minutes)

',
"children": []
},
{
"title": '14. pH Optimization',
"content": '

## 14.1 Solution Preparation

(1) Solution A: Dissolve 30.0 mg of p-nitrophenol palmitate (p-NPP) in 10.00 mL of isopropanol. Store at 4°C in a brown
bottle or a foil-wrapped 15 mL centrifuge tube.

(2) Solution B: Prepare 100 mL of phosphate buffer at each target pH (6-10). Add one drop of Triton X-100 to each, mix well,
and store at 4°C.

## 14.2 Lipase Activity Measurement

(1) Mix 1 mL of Solution A with 9 mL of Solution B (at a specific pH) in a centrifuge tube. Incubate at 37°C for 5 minutes
to form the Reaction Mix.

(2) Add 190 µL of the Reaction Mix to each well of a 96-well plate. Then add:10 µL of enzyme sample, 10 µL of phosphate
buffer at corresponding pH.

(3) Incubate at 37°C for 10 minutes.

(4) Measure the absorbance at 410 nm. Perform each measurement in triplicate and calculate the average value.

(5) Record absorbance values at different pH, and plot an enzyme activity-pH profile.

',
"children": []
},
{
"title": '15. Temperatures Optimization',
"content": '

## 15.1 Solution Preparation

Refer to the previous section for solution preparation. Use a buffer adjusted to the experimentally determined optimal pH.

## 15.2 Lipase Activity Measurement

(1) Add 1 mL of Solution A to 9 mL of Solution B (at optimal pH) in a centrifuge tube. Mix thoroughly and incubate at 37 °C
for 5 minutes to form the Reaction Mix.

(2) Add 190 µL of the Reaction Mix to each well of a 96-well plate. Then add:10 µL of enzyme sample, 10 µL of phosphate
buffe

(3) Incubate at the different temperatures for 10 minutes (20-70 °C).

(4) Measure the absorbance at 410 nm. Perform each measurement in triplicate and calculate the average value.

(5) Record the absorbance values at each temperature and plot an enzyme activity-temperature profile.

',
"children": []
},
] %}
{% block title %}Experiments{% endblock %}
{% block lead %}
![](https://static.igem.wiki/teams/5790/common/experiments.webp)
{% endblock %}
{% block page\_content %}
{% endblock %}

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

To address the global challenge of waste oil recycling and support the enzymatic production of biodiesel—an environmentally
friendly alternative to conventional diesel—we engineered the yeast *Pichia pastoris* (*P. pastoris*)as a robust cellular
factory for high-level production of engineered lipases with enhanced tolerance and catalytic activity. Recognizing that
natural microbial lipases often lack the stability and efficiency required for industrial-scale conversion, we employed a
systematic protein and genetic engineering approach to optimize three key elements: lipase, promoter, and signal peptide.
After selecting the best part, we combined them into a complete lipase production and secretion system, thereby obtaining a
strain with the highest expression level and enzyme activity, which helps to achieve efficient production of lipase and lays
a solid foundation for its application in biodiesel conversion.

',
"children": []
},
{
"title": '1. Screening and Testing of Lipase',
"content": '

Firstly, three promising lipases—CALB (from *Candida antarctica*), GGL (from *Galactomyces geotrichum Y05*), and LipZ01 (from oil-contaminated soil)—were selected for their distinct advantages in activity and stability (**Table 1**).

**Table 1** Three lipase used in this project.

|  |  |  |  |
| --- | --- | --- | --- |
| Name | Source | Genbank | Description |
| CALB | Candida antarctica | [Z30645.1](https://www.ncbi.nlm.nih.gov/nuccore/Z30645.1#tdsub) | One of the most widely studied and applied lipases due to its extensive biocatalytic applications [1]. |
| GGL | Galactomyces geotrichum Y05 | [DQ841279.1](https://www.ncbi.nlm.nih.gov/nuccore/DQ841279.1#tdsub) | Excellent heat resistance and tolerance to organic solvents [2]. |
| LipZ01 | Soil-derived metagenome | [AY304500](https://www.ncbi.nlm.nih.gov/protein/32364181#tdsub) | Good stability over a wide range of temperatures and under alkaline reaction conditions [3]. |

After the company synthesized three lipases that had been optimized for codons, we inserted them into a *P. pastoris* expression vector pPICZαA, which includes the methanol-inducible AOX1 promoter, the α-factor secretion signal, and
a C-terminal His-tag, followed by the AOX1 terminator. The Zeocin resistance gene enables selection in transformants
(**Figure 1A**). We firstly amplified the pPICZαA and three lipase gene, followed by agarose gel
electrophoresis. The results showed the successful amplification of pPICZαA vector (3476 bp), CALB (954 bp), GGL (1692 bp),
and LipZ01 (1428 bp)—with bands corresponding to their expected sizes under UV illumination (**Figure 1B-C**).

![](https://static.igem.wiki/teams/5790/result/1.webp)

**Figure 1** The amplification results of the DNA fragments.

(A) Plasmid diagram. (B-C) Agarose gel electrophoresis result.

Following the amplification of the lipase genes and plasmid vector, the transformation plates confirmed efficient plasmid
introduction into *E. coli* DH5α competent cell (**Figure 2A**). Colony PCR analysis further validated
the presence of the correct insert sizes, with distinct bands corresponding to CALB (1443 bp), GGL (2179 bp), and LipZ01
(1912 bp) observed across multiple clones (**Figure 2B**). Sequencing results confirmed the precise integration
of each expression cassette (**Figure 2C**). These results collectively demonstrate the accurate assembly of
all three plasmids.

![](https://static.igem.wiki/teams/5790/result/2.webp)

**Figure 2** Construction result of three plasmids.

(A) Transformation plates. (B) Colony PCR result. (C)Sequencing result.

The recombinant plasmids pPICZαA-CALB, pPICZαA-GGL, and pPICZαA-LipZ01 were successfully extracted and linearized by
*SacI* enzyme prior to transformation, as confirmed by agarose gel electrophoresis (**Figure 3A**).
Linearizing the plasmid is a critical step because it exposes homologous ends that facilitate targeted integration into the
*P. pastoris* genome via homologous recombination, significantly increasing transformation efficiency compared to
circular plasmids.

Next, we used the lithium chloride transformation method to transfer the linearized plasmid into *P. pastoris*. This
method can generate active yeast cells with permeable cell membranes, enabling the linearized plasmid to smoothly enter the
cells. For the pPICZαA vector, the DNA that has been linearized will specifically integrate into the yeast genome through
homologous recombination at the AOX1 locus in the yeast genome. This process ensures that the target gene can be stably
expressed under the regulation of the inducible AOX1 promoter.

As shown in **Figure 3B,** the linearized plasmids were individually transformed into *P. pastoris* strains, with successful transformants growing on selective YPD plate. The yeast colony PCR results further verified the
correct genomic integration of each lipase gene, showing specific amplification products of 954 bp (CALB), 1692 bp (GGL),
and 1428 bp (LipZ01) (**Figure 3C**). These results collectively demonstrate the successful construction of
three recombinant *P. pastoris* strains capable of expressing the target lipases, providing a solid foundation
for subsequent protein expression and functional studies.

![](https://static.igem.wiki/teams/5790/result/3.webp)

**Figure 3** Construction result of *P. pastoris* containing lipase.

(A) Plasmid Linearization result. (B) Transformation plates. (C) Yeast colony PCR result.

Subsequently, we inoculated the yeast transformants containing the *CALB* gene and activated them in the BMGY medium.
Then, using the BMMY medium, we began to induce the secretion of CALB. **Figure 4** presented the SDS-PAGE
analysis of recombinant lipase secretion in *P. pastoris* after 72-hour fermentation. A distinct protein
band at approximately 44 kDa, corresponding to the expected molecular weight of CALB (43.8 kDa), was clearly observed in the
culture supernatant of all three tested strains at 48 h and 72 h, confirming successful extracellular secretion (**Figure 4A)**. In contrast, no visible bands were detected at the predicted molecular weights for GGL (63.1 kDa) or LipZ01 (51.1 kDa) in
any strain or time point (**Figure 4B-C)**.

The successful secretion of CALB can be attributed to its compatibility with the α-factor secretion signal and efficient
folding in the secretory pathway. The absence of GGL and LipZ01 bands suggests potential issues such as inefficient signal
peptide recognition, protein misfolding leading to endoplasmic reticulum retention, or intracellular degradation. These
results indicate that while the expression system supports CALB secretion, further optimization of the secretion signal or
co-expression of chaperones may be necessary for efficient production of GGL and LipZ01.

![](https://static.igem.wiki/teams/5790/result/4.webp)

**Figure 4** SDS-PAGE results of (A) CALB, (B)GGL, and (C) LipZ01 secretion.

We found that the purity of the lipase secreted into the supernatant was not high enough. To avoid affecting the subsequent
quantitative analysis of the lipase and the enzyme activity test, we carried out nickel column purification on it. Since the
crude supernatant contained impurities that could interfere with accurate quantification and activity assays, we
concentrated it using an ultrafiltration device and exchanged the buffer to PBS (pH 7.4) to optimize binding efficiency.
Following washing and elution steps, a distinct band at approximately 44 kDa, corresponding to CALB, was clearly observed in
the elution fractions (**Figure 5**, E1 and E2), confirming successful purification of the target protein to
high purity. In contrast, no specific bands were detected at the expected molecular weights for GGL (63.1 kDa) or LipZ01
(51.1 kDa) in their respective elution lanes, despite applying the same concentration and purification procedure. This
result is consistent with the earlier secretion data and further confirms that these two lipases were not successfully
secreted in a soluble, His-tagged form, or were present at levels below the detection limit.

![](https://static.igem.wiki/teams/5790/result/5.webp)

**Figure 5** Purification results of three lipase.

Next, we use the BCA method to determine the concentration of the lipase. The BCA method relies on the reduction of Cu²⁺ to
Cu⁺ by peptide bonds in an alkaline medium, with the resulting Cu⁺ ions forming a purple complex with BCA that can be
measured at 562 nm. A series of bovine serum albumin (BSA) standards (25-500 μg/mL) were used to establish a linear
calibration curve, which exhibited excellent linearity with the equation Y = 0.0009982\*X + 0.1326 and a coefficient of
determination (R²) of 0.9934 (**Figure 6**). The high R² value indicateed a strong correlation between
absorbance and protein concentration, validating the reliability of this standard curve for subsequent quantification of
lipase samples.

![](https://static.igem.wiki/teams/5790/result/6.webp)

**Figure 6** The BSA standard curve results of the BCA method

This standardized protocol was applied to determine the concentration of purified lipases in the culture supernatant,
enabling accurate comparison of expression levels among CALB, GGL, and LipZ01 under different experimental conditions. The
BCA assay results quantitatively confirmed the successful secretion of recombinant CALB, which achieved a concentration of
68.21 μg/mL, significantly higher than the negative control (0.96 μg/mL). In contrast, GGL and LipZ01 showed only minimal
concentrations (4.89 and 1.61 μg/mL, respectively), consistent with earlier SDS-PAGE and purification data indicating their
secretion failure (**Figure 7)**. These results validated the effectiveness of the expression and purification
system for CALB, while underscoring the need for further optimization to enable detectable secretion of GGL and LipZ01.

![](https://static.igem.wiki/teams/5790/result/7.webp)

**Figure 7** The quantitative results of the concentration of purified CALB.

We determined the activity of the lipase using the p-nitrophenol method. This assay is based on the hydrolysis of
p-Nitrophenyl esters by lipase, releasing yellow-colored 4-Nitrophenol (4-NP) that absorbs at 450 nm. A series of 4-NP
standards (0–0.1 mmol/L) were prepared, and their absorbance values were measured to establish a linear calibration curve.
The fitted equation Y = 5.924x + 0.01800 exhibited excellent linearity (R² = 0.9857), confirming a strong correlation
between absorbance and 4-NP concentration (**Figure 8)**. This reliable standard curve enables accurate
quantification of lipase activity in subsequent enzymatic assays.

![](https://static.igem.wiki/teams/5790/result/8.webp)

**Figure 8** Standard curve of 4-Nitrophenol

Finally, the lipase activity of the three recombinant strains was measured. The CALB-expressing strain demonstrated the
highest enzymatic activity at 28.59 ± 0.37 U/mL, which was approximately 3.5-fold higher than the activities of GGL (8.15 ±
0.14 U/mL) and LipZ01 (8.41 ± 0.09 U/mL). Statistical analysis (​**​​**​p < 0.0001) confirmed that CALB
activity was significantly higher than both GGL and LipZ01, while no significant difference (ns) was observed between GGL
and LipZ01. The CALB-expressing strain showed significantly higher activity than the negative control (pPICZαA, 5.99 ± 0.70
U/mL), validating the successful expression of it (**Figure 9)**. These results indicate that CALB exhibits
superior catalytic efficiency under the tested conditions.

![](https://static.igem.wiki/teams/5790/result/9.webp)

**Figure 9** Lipase activity test result.

**Summary:**

Based on the successful plasmid construction and yeast transformation of lipases from three different sources, we obtained
the corresponding recombinant strains. Subsequent fermentation and analysis of secretion levels and enzymatic activity
revealed that only CALB was successfully secreted extracellularly, demonstrating measurable enzymatic activity (reaching
28.59 ± 0.37 U/mL). Therefore, in the subsequent optimization phase, we will focus on CALB, specifically targeting the
enhancement of its expression and secretion efficiency through promoter and signal peptide optimization.

',
"children": []
},
{
"title": '2. Promoter and Signal Peptide Optimization',
"content": '

The choice and optimization of promoters in *P. pastoris* are critical for achieving high-level lipase
expression, as promoters directly control transcriptional initiation and strength [4]. Signal peptides are essential for
directing the secretion of heterologous lipases into the extracellular space, simplifying downstream purification and
enhancing yield [5]. Thus, we further screened strong inducible promoters (AOX713, FDH1, FLD1) and efficient secretion
signal peptides (0030α-factor, SWP1-α-factor, KRE1-α-factor), using the classic AOX1 promoter and α-factor signal peptide as
the control, respectively, to maximize transcriptional activation and secretory efficiency (**Table 2**).

**Table 2** Promoters and signal peptides used in this project.

|  |  |  |
| --- | --- | --- |
| Name | Source | Description |
| AOX1  (Control) | Endogenous promoter of *P. pastoris* | The AOX1 promoter is the most widely used methanol-inducible promoter in *P. pastoris* [6]. |
| AOX713 | Rational Design of AOX1 Promoter | Designed by rationally removing inhibitory elements and replicating activating elements [7]. |
| FDH1 | Formate dehydrogenase promoter from *P. pastoris* | This classic NAD⁺-specific FDH catalyzes formate oxidation coupled with NAD⁺ reduction to NADH [8]. |
| FLD1 | Formaldehyde dehydrogenase 1 promoter from *P. pastoris* | Methanol or methylamine induction achieves AOX1-promoter-comparable expression levels [9]. |

**Table 3** Signal peptides used in this project.

|  |  |  |
| --- | --- | --- |
| Name | Source | Description |
| α-factor  (Control) | *S. cerevisiae* | It has a mature receptor-mediated secretion mechanism and is suitable for most exogenous proteins [10]. |
| 0030α-factor | Hybrid signal peptide | Preserves α-factor pro-sequence ER export signaling, 0030 pre-sequence boosts transmembrane translocation [10]. |
| SWP1-α-factor | Hybrid signal peptide | A hybrid signal peptide engineered based on the pro-sequence of the *P. pastoris* SWP1 protein|\'s N-terminal 18 amino acids (MKLFFVGIVTTLLTLLVSC) fused to the α-factor signal peptide [11]. |
| KRE1-α-factor | Hybrid signal peptide | A hybrid signal peptide engineered from the N-terminal 18 amino acids of the *P. pastoris* cell wall synthesis-related protein KRE1 (MLNKLFIAILIVITAVIG) fused to the α-factor signal peptide pro-sequence [11]. |

For the optimization of the promoter, we retained the α-factor signal peptide on the pPIC9K vector and replaced the original
promoter with three candidates: AOX713, FDH1, and FLD1 (**Figure 10A)**. For the optimization of the signal
peptide, we retained the AOX1 promoter on the pPIC9K vector and replaced the signal peptide with three candidates:
0030α-factor, KRE1-α-factor, and SWP1-α-factor (**Figure 10B)**. As shown in the agarose gel electrophoresis
results, the DNA bands for the linearized vectors and the inserted fragments matched the expected sizes, verifying the
successful amplification of all fragments.

![](https://static.igem.wiki/teams/5790/result/10.webp)

**Figure 10** The amplification results of the DNA fragments.

(A)Three promoters. (B) Three signal peptides.

Following the amplification of the different promoters, signal peptides, and plasmid vector, we combined them through
homologous recombination. the transformation plates confirmed efficient plasmid introduction into *E. coli* DH5α
competent cell (**Figure 11**). Colony PCR analysis demonstrated distinct bands of expected sizes: 2188 bp for
AOX713-CALB, 2154 bp for FDH1-CALB, 2169 bp for FLD1-CALB, 2237 bp for AOX1-0030-α-CALB, 2246 bp for AOX1-SWP1-α-CALB, and
2243 bp for AOX1-KRE1-α-CALB. Sequencing results further verified the precise integration of each expression cassette with
correct promoter-signal peptide-CALB organization. These results collectively confirm the accurate assembly of all six
plasmid constructs, providing a reliable foundation for subsequent yeast transformation and protein expression studies.

![](https://static.igem.wiki/teams/5790/result/11.webp)

**Figure 11** The colony PCR and sequencing results of plasmids.

The six recombinant plasmids were then successfully extracted and linearized by *SalI* enzyme before transformation,
as confirmed by agarose gel electrophoresis. Each plasmid displayed a single, clear band after single enzyme digestion,
corresponding to the expected size of the linearized vector (**Figure 12**).

![](https://static.igem.wiki/teams/5790/result/12.webp)

**Figure 12** The linearization results of the plasmids.

After enzyme digestion, the linearized plasmids were transformed into *P. pastoris* competent cell, respectively.
**Figure 13A** showed that transformants of *P. pastoris* growing on selective plates for six
different constructs. Further colony PCR analysis confirmed genomic integration, with distinct bands observed at the
expected size of 954 bp for all constructs when compared to the DNA ladder. These results demonstrated the successful
integration of the *CALB* gene expression cassettes into the yeast genome, validating the effectiveness of the transformation
procedure and providing a reliable foundation for subsequent protein expression studies.

![](https://static.igem.wiki/teams/5790/result/13.webp)

**Figure 13** Construction result of *P. pastoris* containing lipase.

(A) Transformation plates. (B) Yeast colony PCR results.

Next, we conducted fermentation on each of these six strains. For the supernatant from the culture medium that was fermented
for 72 hours in the BMMY medium, we performed SDS-PAGE to determine the secretion of CALB. The SDS-PAGE results showed that
all recombinant strains exhibited distinct protein bands at approximately 43.8 kDa after 72 hours of induction, which were
absent in the pre-induction (0 h) samples and the negative control (NC, pPIC9k empty vector). Notably, the band intensities
varied among strains, with pPIC9k-FDH1-α-CALB and the pPIC9k-AOX1-0030-α-CALB strains demonstrating particularly strong
secretion. These results confirmed the successful secretion of recombinant CALB by all six engineered strains, with varying
expression levels observed under different promoter-signal peptide combinations.

Considering that the CALB purity in the fermentation supernatant of these six strains was relatively high, we did not
perform any additional purification and directly used the BCA kit to determine the concentration. Among promoters, FDH1
yielded the highest CALB concentration (93.82 ± 4.16 μg/mL), significantly outperforming AOX1 (68.21 μg/mL) and FLD1 (61.25
μg/mL). For signal peptides, the 0030-α variant achieved the highest secretion (102.37 μg/mL), markedly surpassing the
standard α-factor (**Figure 14B**). These quantitative data align with band intensities observed in SDS-PAGE,
collectively demonstrating that both promoter strength and signal peptide efficiency critically influence CALB secretion
(**Figure 14A**). The optimal combination of the FDH1 promoter and 0030-α factor signal peptide provides an
effective strategy for enhancing recombinant protein production in *P. pastoris*.

![](https://static.igem.wiki/teams/5790/result/res-14.webp)

**Figure 14** SDS-PAGE verification and quantification of CALB under different promoter regulation and signal
peptide regulation.

Finally, we measured the lipase activity of the six recombinant strains using the p-nitrophenol method.  For the
promoters, the FDH1 promoter yielded the highest enzymatic activity (31.68 U/mL), significantly outperforming AOX1 and FLD1
promoters (​​P ≤ 0.01). The secretion levels of the other two promoters (AOX713 and FLD1) were both lower than that of the
AOX1 promoter (**Figure 15A)**. For the signal peptides, the 0030-α-factor signal peptide led to the highest
activity (31.25 U/mL), while the differences between the other two signal peptides (SWP1-α-factor and KRE1-α-factor) and the
α-factor signal peptide were not statistically significant (ns).

![](https://static.igem.wiki/teams/5790/result/15.webp)

**Figure 15** Lipase activity test results of (A) different promoters and (B) signal peptide.

**Summary:**

In this part, we systematically evaluated the performance of three promoters (AOX713, FDH1, FLD1) and three signal peptides
(0030α-factor, KRE1-α-factor, SWP1-α-factor) for the expression and secretion of CALB in *P. pastoris*. The
recombinant plasmids were successfully constructed and linearized, followed by efficient transformation into *P. pastoris*, as confirmed by colony growth and PCR verification. Fermentation in BMMY medium and subsequent SDS-PAGE analysis revealed
distinct protein bands at approximately 43.8 kDa for all six engineered strains, with varying secretion intensities.
Quantitative BCA assays demonstrated that the FDH1 promoter yielded the highest CALB concentration (93.82 ± 4.16 μg/mL),
while the 0030α-factor signal peptide achieved the highest secretion level (102.37 μg/mL). Enzymatic activity analysis
further confirmed that the FDH1 promoter (31.68 U/mL) and the 0030α-factor signal peptide (31.25 U/mL) delivered superior
lipase activity compared to other variants.

Based on the comprehensive evaluation of protein secretion levels, concentration, and enzymatic activity, the ​**​FDH1 promoter​**​ and ​**​0030α-factor signal peptide​**​ were identified as the most effective components. Consequently, this
optimal combination will be utilized in all subsequent experimental phases to maximize recombinant CALB production.

',
"children": []
},
{
"title": '3. Building and Testing the Optimal Combination',
"content": '

The best-performing combinations were finally integrated into expression cassettes for recombinant strain construction. As
shown in **Figure 16A,** the expression vector pPIC9K was engineered to contain the optimal FDH1 promoter and
0030α-factor signal peptide fused with the *CALB* gene. The agarose gel electrophoresis results (**Figure 16B**)
confirmed successful amplification of both the vector backbone (Amp-FDH1, ~4402 bp) and the insert fragment (0030α-CALB-Kan,
~5954 bp), with bands matching expected sizes. Colony PCR verification (**Figure 16C**) demonstrated successful
transformation of *E. coli* DH5α competent cell, showing specific bands of approximately 2495 bp corresponding to the
target expression cassette in multiple transformants. These results validated the accurate assembly of the optimized
expression system combining the FDH1 promoter and 0030α-factor signal peptide.

![](https://static.igem.wiki/teams/5790/result/16.webp)

**Figure 16** The amplification results of the DNA fragments.

(A) Plasmid diagram. (B) Agarose gel electrophoresis result. (C) Colony PCR and sequencing result.

We then extracted and linearized the correct plasmids for yeast transformation. The agarose gel electrophoresis (**Figure 17**
**A**) confirmed successful linearization, showing a single clear band for the linearized plasmid (lane 2)
compared to the negative control (lane 1). The transformation plates (**Figure 17** **B**)
exhibited robust colony growth, indicating efficient plasmid introduction into *P. pastoris*. Furthermore, yeast
colony PCR analysis (**Figure 17** **C**) validated the genomic integration, revealing a distinct
band at approximately 954 bp corresponding to the FDH1-0030α-CALB expression cassette. These results collectively confirm
the successful construction of the recombinant *P. pastoris* strain.

![](https://static.igem.wiki/teams/5790/result/17.webp)

**Figure 17** Construction result of *P. pastoris* containing lipase**.**

(A) Plasmid Linearization result. (B) Transformation plates. (C) Yeast colony PCR result.

Next, we conducted fermentation on each of these strains (FDH1-0030α-CALB belongs to the experimental group, while
FDH1-α-CALB and AOX1-0030α-CALB constitute the control group) and analyzed CALB secretion. The SDS-PAGE results (**Figure 18A**) clearly showed distinct protein bands at approximately 43.8 kDa in the experimental groups, corresponding to the expected
molecular weight of CALB, while absent in the negative control. Quantitative analysis (**Figure 18B**)
demonstrated that the combination of the FDH1 promoter and 0030-α-factor signal peptide yielded the highest secretion level
(107.02 ± 3.95 μg/mL), significantly outperforming the other constructs (​​P ≤ 0.01). These results confirmed the synergistic
effect of promoter and signal peptide optimization on CALB production.

![](https://static.igem.wiki/teams/5790/result/18.webp)

**Figure 18** SDS-PAGE verification of CALB secretion.

(A) SDS-PAGE result. (B) Quantitative results.

Similarly, we conducted an enzyme activity test. The results showed that compared with the negative control pPIC9K (NC), the
lipase activity of all the recombinant strains was significantly increased. Among them, the combination of the FDH1 promoter
and the 0030-α-factor signal peptide (FDH1-0030-α-factor-CALB) exhibited the highest enzyme activity (33.41 U/mL).
Statistical analysis indicated that the enzyme activity of this combination was significantly higher than that of the
strains that only optimized the promoter (FDH1-α-factor-CALB, 31.68 U/mL) or only optimized the signal peptide
(AOX1-0030-α-factor-CALB, 31.25 U/mL) (P ≤ 0.05), confirming the positive effect of the synergistic optimization of the
promoter and signal peptide on enhancing the catalytic efficiency of the lipase (**Figure 19**).

![](https://static.igem.wiki/teams/5790/result/19.webp)

**Figure 19** Lipase activity test results.

Finally, we investigated the temperature and pH values of the reaction to determine the optimal catalytic conditions of
CALB. As shown in **Figure 20A**, the temperature optimization experiment indicated that CALB exhibited the
highest enzymatic activity (approximately 40 U/mL) at 45°C, while the activity significantly decreased at low temperatures
(25°C) or high temperatures (≥65°C). As shown in **Figure 20B,** the pH optimization results revealed that the
enzyme maintained high activity within the range of 7.5–9.0, with the optimal pH being 8.0. It is noteworthy that CALB
could still maintain over 70% relative activity within a wide pH range (6.0–10.0), indicating its good acid-base stability.
These data clearly defined the optimal reaction conditions of CALB as 45°C and a neutral to weakly alkaline environment,
providing key parameters for its industrial application.

![](https://static.igem.wiki/teams/5790/result/20-new.webp)

**Figure 20** Optimization of (A) reaction temperature and (B) pH for CALB.

**Summary**

In this project, we successfully established an efficient system for recombinant CALB production in *P. pastoris.*
The optimized gene construct, which combines the **FDH1 promoter** and the **0030α factor signal peptide** of the *CALB* gene,
was successfully integrated into the yeast genome. The secretion level of CALB reached 107.02 ± 3.95 μg/mL, and the enzyme
activity was 33.41 U/mL, which were **1.84 times** and **1.17 times** higher than those of the original strain (AOX1-α factor-CALB,
secretion: 68.21 μg/mL, enzyme activity: 28.59 U/mL), respectively. In addition, we determined that **the optimal catalytic
conditions for CALB were 45°C and a pH value of 8.0.** Under these conditions, the enzyme activity could
reach up to 44.96 U/mL, and the enzyme showed high stability within a wide pH range (6.0-10.0). In conclusion,
the collaborative optimization of the FDH1 promoter and the 0030α factor signal peptide is the key strategy for
achieving high-yield active CALB in *P. pastoris.* This recombinant strain and the clearly defined optimal reaction
parameters have laid a solid foundation for the industrial application of this biocatalyst.

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

* **About the Failure of GGL and LipZ01 Secretion**

In the *P. pastoris* system, the failure of GGL and LipZ01 to be successfully secreted into the culture supernatant
may result from multiple interrelated factors. First, the most fundamental reason likely lies in the
**inherent characteristics of proteins**. GGL (63.1 kDa) and LipZ01 (51.1 kDa) possess larger molecular weights
compared to the successfully secreted CALB (43.8 kDa), potentially exhibiting more complex spatial structures. The
endoplasmic reticulum secretory pathway in *P. pastoris* may be unable to effectively recognize or correctly process
the proper folding of these heterologous proteins. This leads to protein retention and aggregation within the endoplasmic
reticulum, ultimately resulting in degradation by intracellular proteasomes [12].

Second, **compatibility between the signal peptide and the target protein** is critical. The α-factor signal
peptide used in this experiment, while effective for many proteins, exhibits efficiency highly dependent on the N-terminal
amino acid sequence of the guided protein. The N-terminal sequences of GGL and LipZ01 may be incompatible with the
recognition and cleavage efficiency of the α-factor signal peptide. This incompatibility prevents the precursor proteins
from being correctly processed by Golgi proteases, such as Kex2, thereby obstructing subsequent secretory steps [13].

* **Effects of Promoters and Signal Peptides on CALB Production**

Different combinations of promoters and signal peptides significantly influence CALB production and secretion levels. This
primarily stems from their regulatory roles in two distinct yet critical processes: gene transcription and protein
transport.

At the transcriptional level, promoter strength directly determines the amount of CALB mRNA transcribed, serving as the
primary determinant of expression levels [14]. For instance, the FDH1 promoter may exhibit stronger constitutive or
uninhibited transcriptional activity than the traditional AOX1 promoter under the fermentation conditions used in this
experiment. This drives greater mRNA synthesis, laying the foundation for high yields.

At the post-translational level, signal peptide efficiency determines whether synthesized CALB protein precursors can be
efficiently processed and transported to the extracellular space. Different signal peptides exhibit varying degrees of
compatibility with the secretory machinery of *P. pastoris*, including endoplasmic reticulum transport, protein
folding and modification, and vesicular transport systems [15]. An efficient signal peptide ensures the CALB protein
precursor is rapidly and accurately translocated into the endoplasmic reticulum lumen, undergoes correct glycosylation and
folding, and is ultimately released into the supernatant via exocytosis. Inefficient signal peptides may cause the protein
precursor to accumulate in the cytoplasm, misfold, or become trapped in the endoplasmic reticulum, leading to degradation.

Furthermore, the physiological state of the host cell represents a deeper underlying cause. High-intensity transcription and
translation driven by strong promoters, coupled with massive protein secretion facilitated by efficient signal peptides,
impose substantial metabolic stress on the host cell. If this stress exceeds the cell\'s tolerance threshold, it can inhibit
cell growth and activate quality control systems like endoplasmic reticulum (ER) stress, leading to extensive protein
degradation [16]. Therefore, the optimal combination (such as the FDH1 promoter paired with the 0030 α-factor signal
peptide) likely strikes the best balance between high expression drive and metabolically tolerable stress, thereby achieving
maximum yield.

',
"children": []
},
{
"title": '5. Future plan',
"content": '

Based on the current experimental results, we have summarized the future project development plan, covering three key
directions: expression system optimization, enzyme immobilization application, and hardware development, in order to further
improve this project.

* **Expression System Optimization**

To address the secretion failure of GGL and LipZ01, we will systematically optimize their expression systems in the future.
Our approach will focus on the following aspects. First, **rationally design or screen signal peptides** to
obtain novel ones with higher compatibility with GGL/LipZ01. Second, we will
**co-express molecular chaperones,** such as endoplasmic reticulum oxidation 1(Ero1) and protein disulfide
isomerase (PDI), to alleviate endoplasmic reticulum stress and promote proper folding of large-molecular-weight lipases.
Concurrently, **rational design of the GGL and LipZ01 genes** (*e.g.*, reducing hydrophobic clusters,
optimizing N-terminal amino acids) will be pursued to enhance their secret ability. Finally, exploration of
protease-deficient host strains (*e.g.*, SMD1163) will be pursued to minimize intracellular degradation.

* **Preparation of Immobilized Lipase**

To enhance lipase stability, reuse efficiency, and reduce application costs, future efforts will focus on developing
immobilization techniques for lipase. The plan involves employing **covalent bonding methods** using carriers
such as amino-silica gel and magnetic nanoparticles, immobilizing purified lipase through crosslinking agents like
glutaraldehyde [17]. This immobilization technique significantly enhances enzyme stability in organic solvents and at
elevated temperatures. Concurrently, we will evaluate encapsulation methods, using sodium alginate-chitosan microspheres or
metal-organic framework (MOF) materials to envelop lipase, creating a protective microenvironment particularly suited for
complex waste oil substrates [18]. We will systematically evaluate the operational half-life and reuse batches of
immobilized enzymes under simulated industrial conditions, aiming to maintain high activity even after continuous use.

![Figure 2](https://static.igem.wiki/teams/5790/result/21.webp)

**Figure 21** Strategies for Lipase Immobilization [19].

* **Test of Conversion of Waste Fats**

We plan to apply the highly active lipase (CALB) developed in our project to test the conversion of waste oils.
Specifically, we will establish a small-scale enzymatic reactor system using restaurant waste oil or gutter oil as
feedstock. Under optimized conditions of temperature (~45°C) and pH (8.0), transesterification reactions will be
conducted to efficiently produce biodiesel (fatty acid methyl esters). We will focus on evaluating the catalytic efficiency,
methanol tolerance, and operational stability of lipases in complex waste oil systems. Additionally, we will explore
enhancing enzyme reuse rates through immobilization techniques, providing data support for subsequent pilot-scale upscaling
and process integration.

![](https://static.igem.wiki/teams/5790/result/22.webp)

**Figure 22** Conversion of Waste Oils Using Immobilized Lipase [20].

Furthermore, we plan to develop a prototype hardware system for enzymatic waste oil conversion (**Figure 23)**.
The core component will be a modular reactor featuring a transparent enzyme chamber packed with immobilized lipase carriers,
allowing real-time observation of the catalytic process. A stainless-steel pre-filtration mesh at the inlet will ensure the
removal of large impurities from waste cooking oil (WCO), while a dedicated glycerol separation layer at the bottom will
enable efficient byproduct recovery. The integrated control unit will regulate key parameters such as temperature, flow
rate, and reaction time to optimize biodiesel yield. This hardware will serve as a demonstrator for scalable, user-friendly
biofuel production, aligning with iGEM\'s goal of converting synthetic biology designs into tangible engineering solutions.

![](https://static.igem.wiki/teams/5790/result/23.webp)

**Figure 23** Hardware system for enzymatic waste oil conversion design in our project.

',
"children": []
},
{
"title": 'Reference',
"content": '

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[8] HU Liyuan, LIU Luyao, BAI Zhonghu,et al.Cofactor specificity engineering and application of formate dehydrogenase from
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[11] Richard Zahrl, Wien (AT); Oezge Ata Aykol, Wien (AT); Diethard Mattanovich,Wien (AT); Brigitte Gasser, Wien (AT)signal
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[13] Wang X, Wang P, Li W, Zhu C, Fan D. Effect and mechanism of signal peptide and maltose on recombinant type III collagen
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[18] Wróblewska-Krepsztul J, Rydzkowski T, Michalska-Pożoga I, Thakur VK. Biopolymers for Biomedical and Pharmaceutical
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[20] Binhayeeding, N., Klomklao, S., Prasertsan, P., & Sangkharak, K. (2020). Improvement of biodiesel production using
waste cooking oil and applying single and mixed immobilised lipases on polyhydroxyalkanoate. Renewable Energy, 162,
1819-1827.

',
"children": []
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