iGEM 2025 — Team ghcis

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{% set table\_data = [{
"title": "",
"content": '

'
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{% block title %}Notebook{% endblock %}
{% block lead %}
notebook
{% endblock %}

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

The primary objective of our project is to address
the global challenge of antibiotic resistance. To achieve this, the GHCIS team optimized the expression of Plectasin
NZ2114—a promising antimicrobial peptide—in Pichia pastoris, aiming to establish a cost-effective and highly efficient
production platform that facilitates the broader application of antimicrobial peptides. Toward this end, we selected
five promoters—AOX1, CAT1, FDH1, and AOX713—and four signal peptides—α-factor, SP4, SP14, and 0030—native regulatory
elements in yeast that enhance both the expression and extracellular secretion of heterologous proteins. For the
active antimicrobial component, we chose Plectasin NZ2114 due to its broad-spectrum antibacterial activity,
particularly its potent efficacy against drug-resistant bacterial strains. Notably, NZ2114 has advanced into clinical
development and demonstrates favorable safety profiles in human studies. Through an iterative engineering workflow, we
constructed recombinant plasmids by fusing NZ2114 with various promoter-signal peptide combinations, followed by yeast
transformation, clone screening, fermentation, and functional validation of NZ2114 expression. Finally, for the most
promising construct, antimicrobial activity was evaluated against Escherichia coli DH5α and Bacillus subtilis,
demonstrating significant growth inhibition and confirming the biological functionality of the expressed peptide.

'
},{
"title": "Cycle 1: Construction of promoter regulatory
elements and expression test of Plectasin NZ2114",
"content": '
',
"children":[{
"title": "Design",
"content": '

We integrated four distinct promoters—AOX1, CAT1,
FDH1, and AOX713—into the pPIC9K backbone plasmid to drive the expression and secretion of the antimicrobial peptide
NZ2114. These promoters function in conjunction with other regulatory elements present in the vector, including signal
peptides, to enable coordinated transcriptional control and efficient protein secretion. In this study, we constructed
a series of recombinant plasmids based on the pPIC9K-AOX1-⍺Factor-NZ2114 framework, in which the AOX1 promoter was
systematically replaced with FDH1, CAT1, or AOX713, resulting in three derivative constructs. Together with the
original AOX1-containing construct, this yielded a total of four promoter variants for comparative evaluation.

![图形用户界面, 应用程序  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/engineer/1.webp)

Fig. 1 Schematic diagram of plasmid design in
Snapgene

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

As shown in Figure 2, during the plasmid construction
process, we first amplified the synthetic target gene fragment by PCR and subsequently purified it to obtain the
desired DNA sequence. Using homologous recombination, we integrated promoter-containing fragments into the pPIC9K
backbone plasmid and transformed the recombinant constructs into Escherichia coli DH5α.

![图片包含 黑暗, 电脑, 亮, 夜晚  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/engineer/2.webp)

Fig .2 DNA electrophoresis of PCR product of promoter
fragment and plasmid backbone fragment.

![图形用户界面  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/engineer/3.webp)

Fig. 3 Transformation of Escherichia coli competent
cells with promoter plasmids and verification of colony monoclonal clones

Colony PCR analysis confirmed successful plasmid
assembly, with amplification of the expected bands observed in multiple clones (Figure 3). Positive colonies were then
selected for large-scale culture and plasmid extraction to prepare for downstream applications.

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

![电脑萤幕画面  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/engineer/4.webp)

Fig. 4 Sanger sequencing was used to identify the
constructed plasmid DNA

Following identification of positive DH5α clones and
plasmid extraction, Sanger sequencing was performed to verify the correct assembly of the recombinant plasmids. As
shown in Figure 4, the sequencing results confirmed accurate integration of the target fragments, thereby validating
the successful construction of the expression vectors.

The recombinant plasmids were successfully
transformed into Pichia pastoris GS115, and following antibiotic selection, engineered yeast strains harboring
distinct promoter constructs were obtained. These transformants were subjected to large-scale cultivation in BMMY
medium with methanol induction to drive the expression and secretion of Plectasin NZ2114 into the culture supernatant.
The presence of the target protein in the supernatants was subsequently analyzed by SDS-PAGE, and as shown in Figure
5, a band corresponding to the expected molecular weight of Plectasin NZ2114 was clearly detected in all engineered
strains, confirming successful expression and extracellular accumulation.

![图片包含 文本  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/engineer/5.webp)

Fig. 5 Expression of plectasin NZ2114 driven by
different promoter and verification by Tris-Tricine PAGE Electrophoresis

The culture supernatants from various yeast
fermentation batches were evaluated for antimicrobial activity against Escherichia coli DH5α using the Oxford cup
diffusion assay. E. coli DH5α, a widely used laboratory strain, served as the indicator organism. All supernatants
derived from strains harboring different promoters exhibited detectable inhibitory effects. Notably, the strain driven
by the AOX713 promoter produced the smallest inhibition zone, indicating relatively lower activity, whereas the
supernatants from strains under the control of AOX1, FDH1, and CAT1 promoters displayed strong and comparable
antibacterial efficacy, as evidenced by similarly sized inhibition zones.

![几个不同颜色的手机  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/engineer/6.webp)

Fig. 6 The inhibition zone experiment was used to
detect the fermentation products of different promoter/signal peptide strain strains.

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

Protein electrophoresis and antimicrobial zone assays
demonstrated that all tested promoter constructs successfully drove the expression of the NZ2114 antimicrobial peptide
in Pichia pastoris, resulting in detectable antibacterial activity. Notably, strains harboring the AOX1, FDH1, and
CAT1 promoters exhibited higher levels of protein expression and stronger inhibitory effects compared to others. Among
these, the AOX1-driven strains displayed superior consistency across biological replicates, indicating enhanced
expression stability. Furthermore, considering the broad applicability and widespread adoption of the AOX1
promoter—particularly in combination with the pPIC9K vector system—it has become a standard choice for recombinant
protein expression in Pichia pastoris. Based on its strong performance, reproducibility, and established utility, AOX1
was selected as a key regulatory element for constructing the high-yield Plectasin NZ2114-producing yeast strain.

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

In this section, we will describe the design of our probiotic-promoter combinations. We will discuss the
genetic constructs we created, including the choice of probiotic strains and the design of the promoter regions.

'
}, {
"title": "Cylcle 2: Construction of signal peptide
regulatory elements and expression test of Plectasin NZ2114",
"content": '

To further screen and evaluate the impact of
different signal peptides on the secretion efficiency of Plectasin NZ2114, we selected four signal peptides—α-factor,
SP4, SP14, and 0030—and fused each to the NZ2114 coding sequence. These fusion constructs were cloned into the pPIC9K
expression vector under the control of its native AOX1 promoter to drive coordinated expression and secretion of the
antimicrobial peptide. In this study, four recombinant plasmids were designed: pPIC9K-AOX1-α-factor-NZ2114, along with
three variants in which the α-factor signal peptide was replaced by SP4, SP14, or 0030, respectively.

![图形用户界面, 文本, 应用程序  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/engineer/7.webp)

Fig. 7 Schematic diagram of the construction of
different signal peptide plasmids in Snapgene

',
"children": [{
"title": 'Build',
"content": '

As shown in the Fig .8, First, based on the
construction of the expression plasmid with the promoter, we chose pPIC9K-AOX1-NZ2114 as the backbone plasmid for
constructing different signal peptide expression plasmids. We obtained gene fragments of the 0030, SP4, and SP14
signal peptide sequences through PCR amplification.

![银色的电脑屏幕  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/engineer/8.webp)

Fig. 8 Gel electrophoresis of agarose nucleic acid
for identifying signal peptide sequence fragments and plasmid backbone fragments

The amplified target fragments were purified and
subsequently ligated into the linearized vector via homologous recombination, followed by transformation into
Escherichia coli DH5α competent cells. Transformants were selected by culturing on solid agar plates, and positive
colonies were screened by colony PCR. As shown in Figure 9, the presence of PCR amplicons of the expected size
confirmed the successful construction of the recombinant plasmids containing different signal peptides. Positive
clones were then expanded in liquid culture, and plasmid DNA was isolated for further use.![图片包含 图形用户界面  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/engineer/9.webp)

Fig. 9 Transformation of signal peptide plasmids into
Escherichia coli competent cells and colony identification

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

Following the isolation of plasmid DNA from
constructs containing different signal peptides, Sanger sequencing was performed. As shown in Figure 10, the
sequencing results confirmed that the obtained sequences precisely matched the intended design, with strong and clean
signals across all reads and no evidence of nucleotide mismatches or mutations.

![图形用户界面  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/engineer/10.webp)

Fig. 10 Sanger sequencing for identifying the plasmid
containing the signal peptide and its construction.

Following successful construction of the signal
peptide-containing plasmid DNA, the plasmids were linearized and transformed into chemically competent GS115 cells.
After screening on selective plates and molecular identification, stable Pichia pastoris transformants harboring
different signal peptide constructs were successfully obtained. These engineered yeast strains were then subjected to
large-scale cultivation and induced for recombinant antimicrobial peptide expression with methanol. Protein
electrophoresis analysis revealed distinct expression levels among the strains, as shown in Figure 11. The
⍺-factor-mediated construct demonstrated significantly higher secretion and expression of the NZ2114 antimicrobial
peptide compared to those directed by SP4, SP14, and 0030 signal peptides.

![图片包含 徽标  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/engineer/11.webp)

Fig. 11 Expression of Plectasin NZ2114 driven by
different signal peptide and Tris-Tricine PAGE Electrophoresis verification

To evaluate the functional efficacy of the secreted
antimicrobial peptides, inhibition zone assays were performed using culture supernatants from Pichia pastoris strains
expressing NZ2114 under the control of different signal peptides. As shown in Figure 12, the supernatant from the
⍺-factor-expressing strain produced a significantly larger inhibition zone compared to those of the SP4, SP14, and
0030 signal peptide strains. In contrast, the strain carrying the 0030 signal peptide exhibited almost no detectable
antibacterial activity.

![几个不同颜色的手机  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/engineer/12.webp)

Fig. 12 The inhibition zone experiment was used to
detect the fermentation products of different promoter/signal peptide strain strains.

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

In this engineering cycle, we constructed and
functionally evaluated engineered yeast strains expressing Plectasin NZ2114 using different signal peptides. Among the
tested variants, the ⍺-factor signal peptide exhibited significantly enhanced secretion efficiency. Based on these
results, ⍺-factor was selected as a key regulatory element for subsequent development of a high-yield Plectasin
NZ2114-producing yeast strain.

'
}]
},{
"title": "Cycle 3: The fermentation and functional
activity testing of high-yield yeast strains",
"content":'',
"children":[{
"title": "Design",
"content": '

Building upon the results from the first two
engineering cycles, small-scale fed-batch fermentations were carried out for the engineered yeast strains harboring
the selected promoter and signal peptide combination. Induced expression was performed under methanol induction, and
two independent batches of fermentation supernatants were collected from both the control strain (empty pPIC9K Pichia
pastoris) and the recombinant strain (pPIC9K-AOX1-⍺-factor-NZ2114 Pichia pastoris). The antimicrobial activity in the
harvested supernatants was comprehensively evaluated to validate the functional efficacy of the target product.

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

Two engineered Pichia pastoris strains were selected
for fermentation: the control strain harboring the empty pPIC9K vector and the recombinant strain expressing NZ2114.
Both strains were initially inoculated into 3 mL YPD medium in test tubes and incubated with shaking. A 1% (v/v)
volume of the resulting seed culture was transferred to 30 mL BMGY medium for overnight growth until the OD600 reached
approximately 4. Cells were then harvested and resuspended in 50 mL BMMY induction medium. Methanol was added every 24
hours to a final concentration of 1% (v/v) to maintain induction of the AOX1 promoter. After 120 hours of induced
expression, cultures were centrifuged, and the supernatants were collected and stored at –80°C for subsequent protein
activity assays.

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

Escherichia coli DH5α and Bacillus subtilis were
selected as model bacterial strains for the subsequent evaluation of antimicrobial peptide activity. The assessment
included growth curve analysis, cell viability assays, and scanning electron microscopy (SEM) examination to
comprehensively characterize the biological effects of the peptide.

**The growth curve assay**

1% (v/v) seed cultures of Escherichia coli and
Bacillus subtilis were inoculated as the initial inoculum and mixed with varying proportions of yeast culture
supernatants. The mixtures were incubated in a shaking incubator, and the OD600 was measured at predetermined time
intervals to monitor bacterial growth.

As shown in Figures 13A and 13B, the E. coli control
group (treated with empty vector supernatant) exhibited normal growth, reaching an OD600 of approximately 1. In
contrast, the NZ2114-containing supernatant significantly suppressed bacterial growth in a dose-dependent manner: 10%
and 25% supplementation resulted in partial inhibition, while 50% and 100% supplementation completely abolished the
growth of E. coli DH5α. Similarly, as depicted in Figures 13C and 13D, B. subtilis in the control group grew robustly,
whereas all NZ2114 treatment groups—except the 0% (no supernatant) control—completely inhibited bacterial growth,
indicating potent and broad-spectrum antimicrobial activity of the expressed peptide.![图形用户界面, 图表, 折线图  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/engineer/13.webp)

Fig. 13 Determination of the inhibitory growth curve
of the culture supernatant of engineered yeast strains: 2h, 4h, 6h, 24h, 30h

**Cell viability assay**

Luminescence signals were quantified using a
microplate reader, and the results are presented in Figure 14. Compared to the control group treated with supernatant
from the empty vector-carrying GS115 yeast strain, the supernatant from the NZ2114-expressing strain significantly
reduced the viability of both Escherichia coli and Bacillus subtilis at 50% concentration. Notably, the inhibitory
effect on Bacillus subtilis was more pronounced, achieving nearly 80% reduction in cell viability.![图表, 条形图  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/engineer/14.webp)

Fig.14 Luminescence detection of control and NZ2114
treated DH5 alpha and B.subtilis in a 50% ratio of fermentation culture medium.

**SEM analysis of bacterial morphological
changes indicative of AMP killing**

To further evaluate the membrane-disrupting activity
and bactericidal efficacy of the antimicrobial peptide NZ2114, Bacillus subtilis was selected as a structurally
relevant model organism for SEM analysis, owing to its shared cellular features with drug-resistant Staphylococcus
aureus.

![](https://static.igem.wiki/teams/5967/engineer/15.webp)

Fig. 15 SEM electron microscope image of Bacillus
subtilis morphology at a 1-micron scale, control group (A, B, C), NZ2114 treatment group (D, E, F)

As shown in Fig. 15, SEM observations at a 1-micron
scale were performed across multiple fields of view. In the control group (Fig. 15 A, B, C), Bacillus subtilis cells
and spores exhibited smooth and intact surface morphologies. In contrast, NZ2114-treated Bacillus subtilis (Fig. 15 D,
E, F) displayed clear morphological alterations, including cell surface wrinkling, collapse, and irregularity. These
structural changes are consistent with the known mechanism of antimicrobial peptides, which exert their bactericidal
effects by disrupting the bacterial membrane. The observed damage provides direct visual evidence that Plectasin
NZ2114 compromises membrane integrity in Bacillus subtilis.

Furthermore, high-resolution SEM imaging at a
200-nanometer scale (Fig. 16) revealed more pronounced ultrastructural damage in the treatment group. Compared to the
uniformly smooth and intact surfaces of control bacteria, NZ2114-treated cells not only exhibited severe wrinkling but
also developed crack-like fissures on their surfaces. These findings further substantiate the membrane-disrupting mode
of action of NZ2114 and confirm its potent bactericidal effect against Bacillus subtilis.

![](https://static.igem.wiki/teams/5967/engineer/16.webp)

Fig. 16 SEM electron microscope image of Bacillus
subtilis morphology at a 200-nanometer scale, control group (A, B), NZ2114 treatment group (D, E)

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

The results from multiple analytical methods
consistently demonstrate that the engineered Pichia pastoris strain developed for high-yield production of Plectasin
NZ2114 effectively expresses and secretes the functional antimicrobial peptide into the culture supernatant,
exhibiting strong antibacterial activity. These findings establish a solid foundation for future industrial-scale
applications. Future efforts should focus on optimizing large-scale fermentation conditions, refining purification
protocols, and evaluating product stability to advance the commercialization of this innovative antimicrobial peptide
in broader therapeutic and biotechnological contexts.

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

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

**Protocol list:**

1. [Preparation of culture medium](https://static.igem.wiki/teams/5967/experiments/1-preparation-of-culture-medium.pdf)
2. [Shaking the bacterial culture](https://static.igem.wiki/teams/5967/experiments/2-shaking-the-bacterial-culture.pdf)
3. [Extract plasmids from bacterial cells](https://static.igem.wiki/teams/5967/experiments/3-extract-plasmids-from-bacterial-cells.pdf)
4. [PCR amplification](https://static.igem.wiki/teams/5967/experiments/4-pcr-amplification.pdf)
5. [Agarose Gel electrophoresis](https://static.igem.wiki/teams/5967/experiments/5-agarose-gel-electrophoresis.pdf)
6. [Gel DNA extraction](https://static.igem.wiki/teams/5967/experiments/6-gel-dna-extraction.pdf)
7. [PCR products purify extraction](https://static.igem.wiki/teams/5967/experiments/7-pcr-products-purify-extraction.pdf)
8. [E. coli transformation](https://static.igem.wiki/teams/5967/experiments/8-e-coli-transformation.pdf)
9. [Solid plate preparation and spread methods](https://static.igem.wiki/teams/5967/experiments/9-solid-plate-preparation-and-spread-methods.pdf)
10. [Pick out monoclonal bacterial](https://static.igem.wiki/teams/5967/experiments/10-pick-out-monoclonal-bacteria.pdf)
11. [Colony identification](https://static.igem.wiki/teams/5967/experiments/11-colony-identification.pdf)
12. [Linearized plasmid of pPIC9K](https://static.igem.wiki/teams/5967/experiments/12-linearized-plasmid-of-ppic9k.pdf)
13. [Clean and purify the restriction enzyme digestion products](https://static.igem.wiki/teams/5967/experiments/13-clean-and-purify-the-restriction-enzyme-digestion-products.pdf)
14. [Preparing competent yeast cells](https://static.igem.wiki/teams/5967/experiments/14-preparing-competent-yeast-cells.pdf)
15. [pPIC9K transformation of GS115](https://static.igem.wiki/teams/5967/experiments/15-ppic9k-transformation-of-gs115.pdf)
16. [Yeast Inducible expression](https://static.igem.wiki/teams/5967/experiments/16-yeast-inducible-expression.pdf)
17. [Oxford Cup antimicrobial test](https://static.igem.wiki/teams/5967/experiments/17-oxford-cup-antimicrobial-test.pdf)
18. [Tris-Tricine SDS PAGE electrophoresis](https://static.igem.wiki/teams/5967/experiments/18-tris-tricine-sds-page-electrophoresis.pdf)
19. [Antimicrobial evaluation of Plectasin NZ2114 peptide by growth curve](https://static.igem.wiki/teams/5967/experiments/19-antimicrobial-evaluation-of-plectasin-nz2114-peptide-by-growth-curve.pdf)
20. [Cell-titer to evaluate the activity of Plectasin NZ2114](https://static.igem.wiki/teams/5967/experiments/20-cell-titer-to-evaluate-the-activity-of-plectasin-nz2114.pdf)
21. [SEM (Scanning Electron Microscope) assay](https://static.igem.wiki/teams/5967/experiments/21-sem-scanning-electron-microscope-assay.pdf)

'
}]%}
{% block title %}Experiments{% endblock %}
{% block lead %}
experiments
{% endblock %}

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

This project employs Pichia pastoris as a host system
to optimize the expression of Plectasin NZ2114 through strategic combinations of promoters and signal peptides. Four
promoters (AOX1, FDH1, CAT1, AOX7₁₃) and four signal peptides (α-factor, SP4, SP14, 0030) were systematically combined
and evaluated to assess their impact on the expression levels of the target protein.

Guided by predefined design objectives, we
constructed a versatile yeast cell factory platform and performed comprehensive expression profiling and functional
validation. By comparing the performance of each engineered strain, we identified the optimal promoter-signal peptide
combination, thereby establishing a robust foundation for scaling up the industrial production of Plectasin NZ2114.

Beyond its applied significance, this study enhances
the mechanistic understanding of how promoters and signal peptides regulate recombinant protein expression in Pichia
pastoris and provides a scalable framework for the efficient production of natural antimicrobial peptides.

'
},{
"title": "Construction of pPIC9K-AOX1-NZ2114,
pPIC9K-FDH1-NZ2114, pPIC9K-CAT1-NZ2114, pPIC9K-AOX713-NZ2114",
"content": '

To evaluate the gene expression driven by different
promoters, we performed genetic construct development based on the pPIC9K plasmid vector. The coding sequence of the
target gene Plectasin NZ2114 and the sequences of various promoters were obtained via gene synthesis, followed by PCR
amplification to generate the desired DNA fragments, as shown in Figure 1.![图片包含 黑暗, 电脑, 亮, 夜晚  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/result/1.webp)

Fig. 1 DNA electrophoresis of PCR product of promoter
fragment and plasmid backbone fragment.

Electrophoretic analysis revealed DNA bands of the
expected size for the target gene fragment at the theoretical position. These bands were documented by imaging and
analyzed for accuracy. The target bands were then excised from the gel and subjected to DNA recovery and purification
for downstream applications. Notably, the AOX713 promoter shares a homologous sequence segment with the AOX1 promoter.
Therefore, we constructed an expression plasmid for NZ2114 driven by AOX713 using the AOX1 plasmid as the backbone.

![图形用户界面  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/result/2.webp)

Fig. 2 Transformation of Escherichia coli competent
cells with promoter plasmids and verification of colony monoclonal clones

Following purification of the target gene fragment,
we employed homologous recombination to assemble the fragment with the pPIC9K plasmid backbone. The ligation mixture
was transformed into competent Escherichia coli cells, and transformants were selected on solid LB agar plates
supplemented with ampicillin. As shown in Figures 2A and 2B, single colonies were successfully obtained. These
colonies were subjected to double digestion analysis and colony PCR to identify clones harboring correctly assembled
plasmids. Positive clones were then scaled up for culture, and plasmid DNA was extracted from the bacterial cultures
and sent to a sequencing service provider for Sanger sequencing to confirm the accuracy of the inserted sequence.

![](https://static.igem.wiki/teams/5967/result/3.webp)

Fig. 3 Sanger sequencing was used to identify the
constructed plasmid DNA

Sequencing results confirmed that 3A corresponds to
pPIC9K-AOX1-NZ2114, 3B to pPIC9K-CAT1-NZ2114, 3C to pPIC9K-FDH1-NZ2114, and 3D to pPIC9K-AOX713-NZ2114. The sequencing
traces of the constructed plasmid vectors exhibited clean single peaks and were fully consistent with the
theoretically designed sequences, confirming accurate assembly of all recombinant plasmids. This successful validation
provides a solid foundation for the subsequent transformation of GS115 competent cells.

![图片包含 红酒, 小, 水, 桌子  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/result/4.webp)

Fig. 4 Construction and identification of engineered
yeast strains for promoters

For the successfully constructed plasmid DNA,
linearization was performed followed by purification of the digested products. The linearized fragments were then
transformed into GS115 chemically competent cells. Transformants were selected by plating on YPD agar supplemented
with G418 and incubated at 30 °C until single colonies appeared. Individual colonies were subjected to colony PCR
using a dedicated microbial lysis buffer for cell lysis, and the resulting lysates were used directly as templates.
PCR products were analyzed by agarose gel electrophoresis. As shown in Figure 4, distinct bands corresponding to the
expected inserts were detected for all promoter-containing plasmids. Positive yeast monoclonal strains were
subsequently expanded in liquid culture to generate seed cultures for downstream experiments.

'
}, {
"title": "Construction of pPIC9K-AOX1-⍺ Factor-NZ2114,
pPIC9K-AOX1-SP4-NZ2114, pPIC9K-AOX1-SP14-NZ2114, pPIC9K-0030-NZ2114",
"content": '

First, based on the construction of the expression
plasmid with the promoter, we chose pPIC9K-AOX1-NZ2114 as the backbone plasmid for constructing different signal
peptide expression plasmids. We obtained gene fragments of the 0030, SP4, and SP14 signal peptide sequences through
PCR amplification. The fragments were analyzed by electrophoresis and matched the theoretical size. Additionally, for
the backbone plasmid, we obtained the linearized pPIC9K backbone plasmid DNA fragment through reverse PCR with
primers.

![银色的电脑屏幕  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/result/5.webp)

Fig. 5 Gel electrophoresis of agarose nucleic acid
for identifying signal peptide sequence fragments and plasmid backbone fragments

The electrophoresis results show that the gene
fragment sequences of the signal peptides are generally small, around 250 bp, and the band positions match the
theoretical sizes. We then proceeded with gel cutting and DNA recovery and purification. Afterward, we obtained
purified gene fragment DNA.

![图片包含 图形用户界面  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/result/6.webp)

Fig. 6 Transformation of signal peptide plasmids into
Escherichia coli competent cells and colony identification

Based on the purified target gene product, we
conducted homologous recombination ligation reactions according to the corresponding concentrations. We then performed
transformation operations on E. coli DH5α competent cells, plated the cells, and successfully obtained monoclonal
cells under cultivation at 37 degrees Celsius. At the same time, we verified the monoclonal cells through colony PCR,
obtaining positive clones in all three plasmid-transformed monoclonal strains. Subsequently, Sanger sequencing
confirmed the successful construction of the plasmid DNA, as shown in the results of Figure 7.

![图形用户界面  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/result/7.webp)

Fig. 7 Sanger sequencing for identifying the plasmid containing the signal peptide and its construction. Figure 7A
shows pPIC9K-AOX1-0030-NZ2114, Figure 7B shows pPIC9K-AOX1-SP4-NZ2114, and Figure 7C shows pPIC9K-AOX1-SP14-NZ2114.
The sequencing signal peaks are stable, and no interference peaks are observed.

Prior to yeast cell transformation, SalI was used to
linearize the constructed plasmid in order to improve transformation efficiency. The digested fragments were purified
and subsequently employed as the linearized DNA for transformation into yeast competent cells.

![图片包含 图形用户界面  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/result/8.webp)

Fig. 8 Construction and identification of signal
peptide engineered yeast strains

As shown in Figure 8, using the successfully
constructed plasmid DNA, we transformed chemically competent GS115 cells. Following selection on YPD solid medium
supplemented with G418, positive colonies were obtained and verified by colony PCR. This resulted in the successful
generation of engineered yeast strains harboring distinct signal peptide expression plasmids.

'
}, {
"title": "Expression level test of Plectasin NZ2114",
"content": '

Building upon engineered yeast strains harboring
different promoter-signal peptide combinations, we performed a preliminary screening. First, the strains were scaled
up for cultivation, followed by fermentation and collection of culture supernatants. The levels of secreted Plectasin
NZ2114 in the supernatants were then analyzed by protein electrophoresis. Given the small molecular weight of the
NZ2114 antimicrobial peptide—only 4.4 kDa—Tris-Tricine SDS-PAGE was employed for analysis and identification. This
system replaces glycine with tricine as the trailing ion, thereby optimizing the buffer composition and significantly
enhancing resolution for low-molecular-weight proteins. By effectively modulating the migration rate of small
proteins, Tris-Tricine electrophoresis prevents their rapid migration through the gel matrix, which can otherwise lead
to poor resolution. Consequently, it has become the method of choice for the separation and detection of proteins and
peptides in the 1–30 kDa range.

![图片包含 文本  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/result/9.webp)

Fig. 9 Expression of plectasin NZ2114 driven by
different promoter and verification by Tris-Tricine PAGE Electrophoresis

As shown in Figure 9, protein expression driven by
different promoters resulted in distinct bands at the theoretical molecular weight of 4.4 kDa for NZ2114. Clear and
well-defined bands were observed with minimal background from non-specific proteins, highlighting the efficiency and
purity advantages of secretory expression in Pichia pastoris. Comparative analysis among promoter groups revealed that
the AOX1 promoter consistently drove high-level and stable expression across multiple clones. The FDH1 and CAT1
promoters achieved protein expression levels comparable to AOX1, whereas the AOX713 promoter exhibited significantly
lower expression. Collectively, these results demonstrate that AOX1, FDH1, and CAT1 are markedly more effective than
AOX713 in mediating recombinant NZ2114 production.

![图片包含 徽标  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/result/10.webp)

Fig. 10 Expression of Plectasin NZ2114 driven by
different signal peptide and Tris-Tricine PAGE Electrophoresis verification

We performed small-scale cultivation and induction of
various Pichia pastoris strains expressing NZ2114 under the control of different signal peptides. Culture supernatants
were collected and analyzed by SDS-PAGE to assess protein secretion efficiency. As shown in Figure 10, the NZ2114 band
intensity was markedly higher in the ⍺-factor group compared to those with SP4, SP14, or 0030 signal peptides. These
results indicate that the ⍺-factor signal peptide is the most effective in mediating the extracellular secretion of
NZ2114 in Pichia pastoris.

'
},{
"title": "Bacteriostatic zone assay",
"content": '

![几个不同颜色的手机  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/result/11.webp)

Fig. 11 The inhibition zone experiment was used to
detect the fermentation products of different promoter/signal peptide strain strains.

Following the evaluation of regulatory element
effects on NZ2114 expression via protein electrophoresis, we observed that the promoters AOX1, FDH1, and CAT1
exhibited comparable expression efficiencies. In contrast, among the signal peptides tested, α-factor demonstrated
markedly superior performance in mediating protein secretion. To further assess the functional impact of these
elements, we evaluated the antimicrobial activity of harvested yeast fermentation supernatants using a phenotypic
assay. As shown in the Oxford cup diffusion assay (Figure 11), the inhibitory activity correlated well with the
expression results: promoter strength followed the order AOX1 ≈ FDH1 ≈ CAT1 > AOX713, while signal peptide efficacy
was ranked as α-Factor > SP4 ≈ SP14 > 0030. Based on these findings and considering both the broad applicability
of the yeast expression system and the reliability of methanol-induced expression, we selected the AOX1 promoter
combined with the α-factor signal peptide as the optimal regulatory pair for high-level production of Plectasin NZ2114
in Pichia pastoris. This combination was subsequently employed in large-scale fermentation and functional
characterization experiments.

'
},{
"title": "The fermentation supernatant of the optimized
GS115 yeast strain: Antibacterial curve test",
"content": '

![图形用户界面, 图表, 折线图  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/result/12.webp)

Fig. 12 Determination of the inhibitory growth curve
of the culture supernatant of engineered yeast strains: 2h, 4h, 6h, 24h, 30h

For the selected promoter and signal peptide
combination, we performed 120-hour fed-batch fermentation of the engineered strain GS115-pPIC9K-AOX1-α Factor-NZ2114
and the control strain GS115-pPIC9K carrying the empty vector. Methanol was supplemented daily at 1% (v/v) to serve as
both an inducer for AOX1-driven expression and a carbon source. Upon completion of fermentation, the culture
supernatants were collected by centrifugation and stored at -80°C for subsequent use. Fresh cultures of Escherichia
coli DH5α and Bacillus subtilis were prepared as indicator strains for antimicrobial activity assays. The
antibacterial assay was carried out in a total volume of 2 mL, with inoculation of 1% (v/v) pre-grown seed culture.
The yeast supernatant was added at varying proportions: 100% (1000 μL), 50% (500 μL), 25% (250 μL), 10% (100 μL), and
0% (0 μL). OD600 was monitored at regular intervals throughout the incubation period. As shown in Figures 12A and 12B,
the E. coli control group (treated with empty vector supernatant) exhibited normal growth, reaching an OD600 of
approximately 1. In contrast, the NZ2114-containing supernatant significantly suppressed bacterial growth in a
dose-dependent manner: 10% and 25% supplementation resulted in partial inhibition, while 50% and 100% supplementation
completely abolished the growth of E. coli DH5α. Similarly, as depicted in Figures 12C and 12D, B. subtilis in the
control group grew robustly, whereas all NZ2114 treatment groups—except the 0% (no supernatant) control—completely
inhibited bacterial growth, indicating potent and broad-spectrum antimicrobial activity of the expressed peptide.

'
},{
"title": "The supernatant from the fermentation of the
optimized GS115 yeast strain: Cell viability test",
"content": '

![图表, 条形图  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5967/result/13.webp)

Fig.13 Luminescence detection of control and NZ2114
treated DH5 alpha and B.subtilis in a 50% ratio of fermentation culture medium.

Based on the antibacterial growth curve analysis, we
performed a cell viability assay on bacterial cells treated with 50% fermentation supernatant. Cell viability was
assessed using the CellTiter-Glo luminescent assay, which involves cell lysis followed by a chemiluminescent reaction.
Luminescence signals were quantified using a microplate reader, and the results are presented in Figure 13. Compared
to the control group treated with supernatant from empty vector-carrying GS115 yeast, the supernatant from the
NZ2114-expressing strain significantly reduced the viability of both Escherichia coli and Bacillus subtilis at the 50%
concentration. Notably, the inhibitory effect on Bacillus subtilis was more pronounced. These findings further confirm
that our engineered Pichia pastoris GS115 strain successfully produces functional Plectasin NZ2114, leading to
effective suppression of bacterial cell viability and growth.

'},{
"title": "The supernatant from the optimized yeast
strain: SEM analysis of bacterial morphological changes indicative of killing",
"content": '

Given that this study aims to address the clinical
challenge of drug-resistant Staphylococcus aureus, Bacillus subtilis was selected as the primary model organism for
scanning electron microscopy (SEM) analysis to evaluate the bactericidal efficacy of NZ21114. Both Bacillus subtilis
and Staphylococcus aureus are Gram-positive bacteria, yet they differ morphologically—rod-shaped (bacilli) versus
spherical (cocci). In contrast, Escherichia coli, a Gram-negative bacterium, exhibits more pronounced differences in
cell wall structure and overall biological characteristics compared to Staphylococcus aureus. Therefore, Bacillus
subtilis serves as a phylogenetically and structurally relevant surrogate that allows for meaningful assessment of
antimicrobial effects while maintaining experimental relevance to the target pathogen.

![](https://static.igem.wiki/teams/5967/result/14.webp)

Fig. 14 SEM electron microscope image of Bacillus
subtilis morphology at a 1-micron scale, control group (A, B, C), NZ2114 treatment group (D, E, F)

As shown in Fig. 14, SEM observations at a 1-micron
scale were performed across multiple fields of view. In the control group (Fig. 14 A, B, C), Bacillus subtilis cells
and spores exhibited smooth and intact surface morphologies. In contrast, NZ2114-treated Bacillus subtilis (Fig. 14 D,
E, F) displayed clear morphological alterations, including cell surface wrinkling, collapse, and irregularity. These
structural changes are consistent with the known mechanism of antimicrobial peptides, which exert their bactericidal
effects by disrupting the bacterial membrane. The observed damage provides direct visual evidence that Plectasin
NZ2114 compromises membrane integrity in Bacillus subtilis.

Furthermore, high-resolution SEM imaging at a
200-nanometer scale (Fig. 15) revealed more pronounced ultrastructural damage in the treatment group. Compared to the
uniformly smooth and intact surfaces of control bacteria, NZ2114-treated cells not only exhibited severe wrinkling but
also developed crack-like fissures on their surfaces. These findings further substantiate the membrane-disrupting mode
of action of NZ2114 and confirm its potent bactericidal effect against Bacillus subtilis.

![](https://static.igem.wiki/teams/5967/result/15.webp)

Fig. 15 SEM electron microscope image of Bacillus
subtilis morphology at a 200-nanometer scale, control group (A, B), NZ2114 treatment group (D, E)

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