iGEM 2025 — Team ucs-x

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

**Team Calendar 2024**

**December**

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Event/Date | 9th |  |  |  |  | 28th | 31st |
| Recruiting of new members |  |  |  |  |  |  |  |
| Online members\' training (theory & practical) |  |  |  |  |  |  |  |
| Project brainstorm |  |  |  |  |  |  |  |

**Team Calendar 2025**

**January**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Event/Date | 1st | 2nd |  | 4th |  |  | 11th |  |  | 18th |  |  | 25th |  | 31st |
| Online members\' training (theory & practical) |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Project brainstorm |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Project kick-off meeting |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

**February**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Event/Date |  |  | 5th | 6th | 7th | 8th |  |  |  |  |  |  |  |  | 28th |
| Construction of plasmids |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Human Practice |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Members\' training (theory & practical) |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| wiki development |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

**March**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Event/Date |  |  |  | 15th |  |  |  |  |  |  |  |  |  | 29th | 31st |
| Online members\' training (theory & practical) |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| wiki development |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Modeling |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

**April**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Event/Date |  |  | 5th |  |  | 12th |  |  | 20th |  |  | 25th |  |  | 30th |
| Online members\' training (theory & practical) |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| wiki development |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Modeling |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

**May**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Event/Date | 1st |  |  |  |  |  |  |  |  |  |  |  |  |  | 31st |
| wiki development |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

**June**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Event/Date | 1st |  |  |  |  |  |  | 23th | 24th | 25th | 26th | 27th | 28th | 29th | 30th |
| Construction of plasmids |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Expression and purification of enzymes |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Functional identification |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| wiki development |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Human Practice |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Modeling |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

**July**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Event/Date | 1st |  |  | 6th |  |  | 10th |  |  |  |  |  |  |  | 31st |
| wiki development |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Modeling |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

**August**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Event/Date | 1st |  |  | 5th |  |  |  |  |  |  | 20th |  |  |  | 31st |
| wiki development |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| wiki content improvement |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

**September**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Event/Date | 1st |  |  |  |  |  | 14th |  |  | 20th |  |  |  |  | 30th |
| UI website design |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| coding modification |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

**October**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Event/Date | 1st |  |  | 5th |  |  |  |  | 12th |  |  |  |  |  | 31st |
| Integration of the PPT for the presentation |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Members are training for the presentation |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

'}]%}
{% block title %}Notebook{% endblock %}
{% block lead %}
![](https://static.igem.wiki/teams/5764/header/v1/notebook.webp)
{% endblock %}

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

Papaya potion conducted a total of five repeats of
the DBTL cycle and successfully synthesized the cysteine protease papain along with four of its isoenzymes.

![1](https://static.igem.wiki/teams/5764/engineering/1.webp)

**Figure 1. The Flow of our plan**

**1. Design:** Research and
identification of our isoenzyme-coding genes, along with selection of a competent vector and competent host bacterial
cells.

**2. Build:** We obtained the gene
sequences from the NCBI database and codon-optimized the sequences for E. coli expression to synthesize our enzymes.

**3. Test:** Conducted a series of
protein tests to experiment with the compatibility and activity of our five enzymes in order to come up with the best
combination of enzymes.

**4. Learn:** Learning from the results
and using molecular docking to explain the mechanisms in which our enzyme combinations turned out to rank as
accordingly.

'
},{
"title": "Recombinant plasmid construction",
"content": '',
"children":[{
"title": "Design-Build-Test-Learn cycle",
"content": '

The objective is to enhance skincare formulations
through the synergistic actions of isoenzymes of the cysteine protease Papain, known for its exfoliating and
moisturizing properties. Isoenzymes of Papain were designed with the goal of optimizing enzyme activity for skincare
under a broad range of skin types and pH conditions. Papain was selected as the model enzyme based on its ability to
catalyze the breakdown of peptide bonds within keratin proteins located in the stratum corneum, the outermost layer of
the epidermis. Papain has a good hydrolyzing effect on many kinds of proteins, reducing the connection between
cornified desmosomes. Therefore, papain can act on the aging stratum corneum of the skin, promoting its decomposition
and degradation, and removing it to achieve the effect of skin rejuvenation and promote cell growth. It is also
gentler than traditional acid peeling. NCBI BLAST was used to identify genes that code for isoenzymes of the cysteine
protease Papain, the reference enzyme. The criterion was that if any gene shared a base sequence similarity of greater
than 50% and less than 80% with Papain, it qualifies as an isoenzyme’s gene because anything above 80% is regarded as
being the same as Papain. Following this, AlphaFold was used to predict the structure of the isoenzymes and select the
most stable ones. Finally, the five isoenzymes selected were cysteine protease XCP2, Cicer arietinum; cysteine
protease XCP2, Abrus precatorius; cysteine protease XCP2, Actinidia eriantha; cysteine protease XCP2, Cajanus cajan;
and Papain.

![](https://static.igem.wiki/teams/5764/engineering/2.webp)

**Figure 2. Recombinant plasmid construction**

'
}]
}, {
"title": "Cycle 1: pET28a-Papain BBa\_25ULT0GX",
"content": '

**Design:**

To express the cysteine protease papain for our
skincare formulation, the pET28a-papain recombinant plasmid was constructed by first obtaining the isoenzyme gene
sequence from the NCBI database. The sequence was codon-optimized for *E.coli* expression and amplified using
PCR to produce adequate quantities for downstream cloning. The expression plasmid used was pET28a, a conventional
vector in molecular biology research due to its high-level expression capabilities and His-tag purification
compatibility. The isoenzyme coding sequence was placed into the pET28a vector directly downstream of the T7 promoter
and a synthetic ribosome binding site, enabling high-level transcription by T7 RNA polymerase. A lac operator element
was placed between the promoter and the papain gene to allow LacI-mediated repression that is relieved upon IPTG
addition. When IPTG is added to a bacterial culture, it binds to the lac repressor, preventing the repressor from
binding to the lac operator, and thus allowing the expression of genes under the control of the lac promoter. The
plasmid also confers kanamycin resistance, allowing for selection in *E.coli* cells.

![](https://static.igem.wiki/teams/5764/engineering/3.webp)

**Figure 3. The map of pET28a plasmid**

The pET28a-papain construct enables us to tightly
control and efficiently express papain under laboratory conditions. Homologous recombination method was used for the
construction of pET28a-papain recombinant plasmids rather than using DNA ligase because of its greater efficiency and
accuracy in combining the Papain gene with pET28a.

![](https://static.igem.wiki/teams/5764/engineering/4.webp)

**Figure 4. The map of pET28a-papain**

**Build:**

The pET-28a-papain construct is designed to express
the Carica papaya papain protease in *E. coli* under the control of the T7 promoter, enabling high-level,
inducible protein expression. The system utilizes the lac operon-based regulation, where IPTG induction activates T7
RNA polymerase, initiating transcription of the papain coding sequence. A His-tag is fused to the protein to
facilitate purification via affinity chromatography. This design enables efficient production and downstream
application of recombinant papain for functional assays such as protein degradation or skin exfoliation.

The papain gene fragment (678 bp) was amplified using
PCR, and both the gene and the pET-28a(+) vector were digested with NdeI and HindIII restriction enzymes to generate
compatible sticky ends. Figure 5 shows the agarose gel electrophoresis results of PCR. We can find out that the DNA
fragment of Papain is approximately 678 bp in length, which is consistent with the predicted size. This indicates that
the target gene was successfully amplified.

![papain扩增](https://static.igem.wiki/teams/5764/engineering/5.webp)

**Figure 5. Gel electrophoresis of PCR amplified papain gene**

The purified insert and backbone were then ligated
using Homologous recombination enzyme. The ligation product was transformed into *E. coli* BL21(DE3) competent
cells via heat shock to obtain the recombinant plasmid. After transformation, the cells were recovered at 37°C for 1
hour, plated on LB agar containing kanamycin, and incubated overnight. Colonies were picked, and the plasmid was
extracted and verified by gel electrophoresis and Sanger sequencing. After homologous recombination, the recombinant
plasmids were transformed into chemically competent *E. coli* BL21(DE3) cells via the heat shock method.
Following overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies
were observed (Figure 6A), indicating successful transformation. To verify the presence of the target gene, five
individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 6B),
each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion
of the target gene. To confirm the accuracy of plasmid construction, recombinant pET28a plasmids were sent to a
commercial sequencing facility for Sanger sequencing. Sequence alignment (Figure 6C) showed that the inserted gene
fragments matched the designed sequences exactly, with no frameshift mutations or base substitutions, indicating
successful and accurate cloning of the target genes.

![Pet-papain组合图_画板 1](https://static.igem.wiki/teams/5764/engineering/6.webp)

**Figure 6.** **Colony growth, gel electrophoresis result and DNA sequencing
of pET28a-papain**

**Test:**

Protein expression was induced with IPTG (1 mM) in
*E. coli* BL21(DE3), followed by cell lysis using a sonicator under cold conditions to preserve enzyme
activity. The recombinant papain protein was purified using nickel affinity chromatography via its 6×His-tag. The
purification process involved washing with low (buffer A) and high (buffer B) concentrations of imidazole. The
collected fractions were analyzed by SDS-PAGE. As shown in Figure 7, distinct protein bands were observed in all
lysate samples. A ~24 kDa band was detected in the papain-expressing strain, consistent with the expected molecular
weight of papain. These results confirmed that XCP2-papain recombinant enzymes were successfully expressed in *E.
coli*.

![XCP2-papain 蛋白图](https://static.igem.wiki/teams/5764/engineering/7.webp)

**Figure 7. SDS-PAGE results of pET28a-Papain**

**Learn:**

Before we start the experiment, we used AlphaFold to
predict their three-dimensional structures. The predicted structure provides a solidfoundation for the subsequent
molecular docking experiments.That’s the molecular docking results. According to this table, we can reach the first
conclusion: the binding force stability values of each isoenzyme are different. Obviously, the negative value of
papain is the largest. It indicates that its binding affinity is the strongest.Besides, we may find from Table 1,
papain doesn’t only form s hydrogen bonds with substrates but also generates additional π bonds, which can enhance
binding stability. It explains the docking results. In our ELISA test, we found that papain has the highest
concentration among the 5 isoenzymes which proves the prediction.

**Table 1 Indication of the binding affinity
between five enzymes and the substrate**

![](https://static.igem.wiki/teams/5764/engineering/table1.webp)

'}, {
"title": "Cycle 2: pET28a-Abrus Precatorius BBa\_25ZPQ33J",
"content": '

**Design:**

The design is pragmatically like that of cycle 1,
except we used XCP2-Abrus Precatorius, an isoenzyme of papain, in place of papain as it may show greater enzyme
activity either individually or when in synergy with other isoenzymes.

![](https://static.igem.wiki/teams/5764/engineering/8.webp)

**Figure 8. The map of pET28a-Abrus precatorius**

**Build:**

The pET-28a-XCP2-Abrus precatorius construct is
engineered to express the XCP2 cysteine protease derived from Abrus precatorius in *E.coli*. The gene is placed
downstream of the T7 promoter, enabling tightly controlled and high-yield expression upon IPTG induction via the lac
operon system. The vector includes an N-terminal His-tag for simplified purification using nickel affinity
chromatography. This design allows for efficient production of functional XCP2 protease to evaluate its
keratin-degrading and exfoliation potential in vitro.

The XCP2-Abrus gene (1083 bp), a cysteine protease
from Abrus precatorius, was amplified by PCR. Both the PCR product and the pET-28a(+) vector were digested with NdeI
and HindIII to generate compatible ends. Figure 9 shows the agarose gel electrophoresis results of PCR. We can find
out that the DNA fragment of XCP2-Abrus is approximately 1083 bp in length, which is consistent with the predicted
size. This indicates that the target gene was successfully amplified.

![Abr 扩增](https://static.igem.wiki/teams/5764/engineering/9.webp)

**Figure 9. Agarose gel electrophoresis results of PCR for XCP2-Abrus**

The purified insert and backbone were then ligated
using Homologous recombination enzyme. The ligation product was transformed into *E.coli* BL21(DE3) competent
cells via heat shock to obtain the recombinant plasmid. After transformation, the cells were recovered at 37°C for 1
hour, plated on LB agar containing kanamycin, and incubated overnight. Colonies were picked, and the plasmid was
extracted and verified by gel electrophoresis and Sanger sequencing. After homologous recombination, the recombinant
plasmids were transformed into chemically competent *E.coli* BL21(DE3) cells via the heat shock method.
Following overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies
were observed (Figure 10A), indicating successful transformation. To verify the presence of the target gene, five
individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 10B),
each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion
of the target gene. To confirm the accuracy of plasmid construction, recombinant pET28a plasmids were sent to a
commercial sequencing facility for Sanger sequencing. Sequence alignment (Figure 10C) showed that the inserted gene
fragments matched the designed sequences exactly, with no frameshift mutations or base substitutions, indicating
successful and accurate cloning of the target genes.

![Pet-Abr组合图_画板 1](https://static.igem.wiki/teams/5764/engineering/10.webp)

**Figure 10. Colony growth, gel electrophoresis result and DNA sequencing of pET28a-XCP2-Abrus**

**Test:**

Protein expression was induced using 1 mM IPTG. Cells
were lysed by sonication at low temperature. The recombinant XCP2-Abrus protein was purified using nickel affinity
chromatography. The washing steps included buffer A (low imidazole) and buffer B (high imidazole), followed by
SDS-PAGE analysis of elution fractions.Verified clones were transformed into *E. coli* BL21(DE3) for protein
expression. As shown in Figure 11, distinct protein bands were observed in lysate samples. A ~39 kDa band was detected
in the Abrus-expressing strain, consistent with the expected molecular weight of XCP2-Abrus. These results confirmed
that recombinant enzymes were successfully expressed in *E. coli*.

![XCP2-Abrus precatorius 蛋白图](https://static.igem.wiki/teams/5764/engineering/11.webp)

**Figure 11. SDS-PAGE results of pET28a-XCP2-Abrus**

**Learn:**

Abr forms hydrogen bond with substrates which leads
to a high stability. However, it has less affinity caused by its different binding sites. The prediction is proved by
our ELISA test. As the same problem as Act, we didn’t succeed to transform the recombinate plasmid, but we improve the
plasmid concentration.

'},{
"title": "Cycle 3: pET28a-Actinidia Eriantha
BBa\_25TUUN1F",
"content": '

**Design:**

The design is pragmatically like that of cycle 1,
except we used XCP2-Actinidia Eriantha, an isoenzyme of papain, in place of papain as it may show greater enzyme
activity either individually or when in synergy with other isoenzymes.

![](https://static.igem.wiki/teams/5764/engineering/12.webp)

**Figure 12. The map of pET28a-Actinidia Eriantha**

**Build:**

The pET-28a-XCP2-Actinidia eriantha construct is
designed to express the XCP2 cysteine protease derived from Actinidia eriantha (hardy kiwifruit) in E. coli under the
control of the T7 promoter. The system employs IPTG-inducible lac operon regulation, ensuring tightly controlled and
high-level protein expression. A 6×His-tag is fused to the N-terminus to enable convenient purification via nickel
affinity chromatography. This recombinant expression system facilitates functional studies of XCP2’s proteolytic
activity and its potential applications in keratin degradation and exfoliation.

The XCP2-like gene from Actinidia eriantha (1089 bp)
was PCR-amplified and digested along with the pET-28a(+) vector using NdeI and HindIII. After purification, the
digested fragments were ligated using Homologous recombination enzyme. Figure 13 shows the agarose gel electrophoresis
results of PCR. We can find out that the DNA fragment of XCP2-Actinidia is approximately 1089 bp in length, which is
consistent with the predicted size. This indicates that the target gene was successfully amplified.

![Act 扩增](https://static.igem.wiki/teams/5764/engineering/13.webp)

**Figure 13. Agarose gel electrophoresis results of PCR for XCP2-Actinidia**

The purified insert and backbone were then ligated
using Homologous recombination enzyme. The ligation product was transformed into *E.coli* BL21(DE3) competent
cells via heat shock to obtain the recombinant plasmid. After transformation, the cells were recovered at 37°C for 1
hour, plated on LB agar containing kanamycin, and incubated overnight. Colonies were picked, and the plasmid was
extracted and verified by gel electrophoresis and Sanger sequencing. After homologous recombination, the recombinant
plasmids were transformed into chemically competent *E.coli* BL21(DE3) cells via the heat shock method.
Following overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies
were observed (Figure 14A), indicating successful transformation. To verify the presence of the target gene, five
individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 14B),
each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion
of the target gene. To confirm the accuracy of plasmid construction, recombinant pET28a plasmids were sent to a
commercial sequencing facility for Sanger sequencing. Sequence alignment (Figure 14C) showed that the inserted gene
fragments matched the designed sequences exactly, with no frameshift mutations or base substitutions, indicating
successful and accurate cloning of the target genes.

![Pet-Act组合图_画板 1](https://static.igem.wiki/teams/5764/engineering/14.webp)

**Figure 14. Colony growth, gel electrophoresis result and DNA sequencing of pET28a-XCP2-Actinidia**

**Test:**

The verified plasmid was transformed into *E.coli*BL21(DE3) for protein expression. Following IPTG induction, the cells were harvested and lysed with sonication at
low temperature. The recombinant protein was purified using Ni-NTA affinity chromatography, including stepwise washes
with imidazole buffers. As shown in Figure 15, distinct protein bands were observed in lysate samples. A ~39 kDa band
was detected in the Actinidia-expressing strain, consistent with the expected molecular weight of XCP2-Actinidia.
These results confirmed that recombinant enzymes were successfully expressed in *E.coli*.

![XCP2-Actinidia eriantha 蛋白图](https://static.igem.wiki/teams/5764/engineering/15.webp)

**Figure 15. SDS-PAGE result of pET28a-XCP2-Actinidia**

**Learn:**

Actinidia has the second largest affinity. We may
find that it forms hydrogen bond with substrates. The difference of affinity to other isoenzymes is caused by the
different binding sites. In the ELISA test, we proved its high efficiency. However,we made some mistakes in the
process of heat shock transformation of recombinate plasmid of Act to E. Coli(DH5α) in the first time, but in the
second time, we succeeded and increased the concentration of the plasmid.

'
},{
"title": "Cycle 4: pET28a-Cajanus Cajan BBa\_25FRHJRN",
"content": '

**Design:**

The design is pragmatically like that of cycle 1,
except we used XCP2-Cajanus Cajan, an isoenzyme of papain, in place of papain as it may show greater enzyme activity
either individually or when in synergy with other isoenzymes.

![](https://static.igem.wiki/teams/5764/engineering/16.webp)

**Figure 16. The map of pET28a-Cajanus Cajan**

**Build:**

The pET-28a-XCP2-Cajanus cajan construct is
engineered to express the XCP2 cysteine protease derived from Cajanus cajan (pigeon pea) in E. coli under the control
of the T7 promoter. The system utilizes the lac operon for IPTG-inducible expression, enabling tight regulation and
high yield of the recombinant protein. An N-terminal His-tag is incorporated to facilitate purification through nickel
affinity chromatography. This construct allows for efficient production and activity analysis of XCP2, with the goal
of evaluating its keratin-degrading and exfoliation potential for skincare-related applications.

The XCP2-Cajanus gene (1083bp) was obtained by PCR
amplification from a codon-optimized synthetic template. Both the insert and pET-28a(+) backbone were digested with
NdeI and HindIII to create sticky ends. The digested fragments were ligated using Homologous recombination enzyme to
generate the plasmid pET28a-XCP2-Cajanus. Figure 17 shows the agarose gel electrophoresis results of PCR. We can find
out that the DNA fragment of XCP2-Cajanus is approximately 1083 bp in length, which is consistent with the predicted
size. This indicates that the target gene was successfully amplified.

![CAJ 扩增](https://static.igem.wiki/teams/5764/engineering/17.webp)

**Figure 17. Agarose gel electrophoresis results of PCR for XCP2-Cajanus**

The purified insert and backbone were then ligated
using Homologous recombination enzyme. The ligation product was transformed into *E.coli* BL21(DE3) competent
cells via heat shock to obtain the recombinant plasmid. After transformation, the cells were recovered at 37°C for 1
hour, plated on LB agar containing kanamycin, and incubated overnight. Colonies were picked, and the plasmid was
extracted and verified by gel electrophoresis and Sanger sequencing. After homologous recombination, the recombinant
plasmids were transformed into chemically competent *E.coli* BL21(DE3) cells via the heat shock method.
Following overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies
were observed (Figure 18A), indicating successful transformation. To verify the presence of the target gene, five
individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 18B),
each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion
of the target gene. To confirm the accuracy of plasmid construction, recombinant pET28a plasmids were sent to a
commercial sequencing facility for Sanger sequencing. Sequence alignment (Figure 18C) showed that the inserted gene
fragments matched the designed sequences exactly, with no frameshift mutations or base substitutions, indicating
successful and accurate cloning of the target genes.

![Pet-CAJ组合图_画板 1](https://static.igem.wiki/teams/5764/engineering/18.webp)

**Figure 18. Colony growth, gel electrophoresis result and DNA sequencing of pET28a-XCP2-Cajanus**

**Test:**

The confirmed plasmid was transformed into*E.coli* BL21(DE3) for protein expression. IPTG was added to induce expression, and the culture was harvested
after 20 hours. Cell lysis was performed using a sonicator under low-temperature conditions to prevent protease
denaturation. Purification was carried out using nickel affinity chromatography. The column was washed with buffer A
(low imidazole) and eluted with buffer B (high imidazole). Collected fractions including lysate, flow-through, and
elution were analyzed via SDS-PAGE. As shown in Figure 19, distinct protein bands were observed in lysate samples. A
~39 kDa band was detected in the Cajanus-expressing strain, consistent with the expected molecular weight of
XCP2-Cajanus. These results confirmed that recombinant enzymes were successfully expressed in *E.coli*.

![XCP2-Cajanus cajan 蛋白图](https://static.igem.wiki/teams/5764/engineering/19.webp)

**Figure 19. SDS-PAGE result of pET28a-XCP2-Cajanus**

**Learn:**

Similar to other isoenzymes, Caj forms hydrogen bond
with substrates which leads to a high stability. However, it has less affinity caused by its different binding sites.
The prediction is proved by our ELISA test.

'
},{
"title": "Cycle 5: pET28a-Cicer Arietinum BBa\_251N76W5",
"content": '

**Design:**

The design is pragmatically like that of cycle 1,
except we used XCP2-Cicer Arietinum, an isoenzyme of papain, in place of papain as it may show greater enzyme activity
either individually or when in synergy with other isoenzymes.

![](https://static.igem.wiki/teams/5764/engineering/20.webp)

**Figure 20. The map of pET28a-Cicer Arietinum**

**Build:**

The pET-28a-XCP2-Cicer arietinum construct is
designed to express the cysteine protease XCP2 from Cicer arietinum (chickpea) in *E.coli*. The coding sequence
is placed under the control of the T7 promoter, allowing IPTG-inducible expression via the lac operon. An N-terminal
His-tag is included to facilitate protein purification using nickel affinity chromatography. This design enables
efficient recombinant production of XCP2 for functional assays, with the goal of evaluating its keratin-degrading
ability and potential use in skin exfoliation or biological material recycling.

The XCP2-like Cicer gene (1095bp) was PCR-amplified
from a synthesized, codon-optimized template. The gene fragment and pET28a(+) plasmid were digested with NdeI and
HindIII to generate sticky ends. Using Homologous recombination enzyme, we ligated the insert into the linearized
vector to form the recombinant plasmid pET28a-XCP2-Cicer. Figure 21 shows the agarose gel electrophoresis results of
PCR. We can find out that the DNA fragment of XCP2-Cicer is approximately 1095bp in length, which is consistent with
the predicted size. This indicates that the target gene was successfully amplified.

![cic 扩增](https://static.igem.wiki/teams/5764/engineering/21.webp)

**Figure 21. Agarose gel electrophoresis results of PCR for XCP2-Cicer**

The purified insert and backbone were then ligated
using Homologous recombination enzyme. The ligation product was transformed into *E.coli* BL21(DE3) competent
cells via heat shock to obtain the recombinant plasmid. After transformation, the cells were recovered at 37°C for 1
hour, plated on LB agar containing kanamycin, and incubated overnight. Colonies were picked, and the plasmid was
extracted and verified by gel electrophoresis and Sanger sequencing. After homologous recombination, the recombinant
plasmids were transformed into chemically competent *E.coli* BL21(DE3) cells via the heat shock method.
Following overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies
were observed (Figure 22A), indicating successful transformation. To verify the presence of the target gene, five
individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 22B),
each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion
of the target gene. To confirm the accuracy of plasmid construction, recombinant pET28a plasmids were sent to a
commercial sequencing facility for Sanger sequencing. Sequence alignment (Figure 22C) showed that the inserted gene
fragments matched the designed sequences exactly, with no frameshift mutations or base substitutions, indicating
successful and accurate cloning of the target genes.

![Pet-CIC组合图_画板 1_画板 1](https://static.igem.wiki/teams/5764/engineering/22.webp)

**Figure 22. Colony growth, gel electrophoresis result and DNA sequencing of pET28a-XCP2-Cicer**

**Test:**

The verified plasmid was transformed into *E.coli*BL21(DE3). IPTG was added to induce expression of the XCP2-Cicer protein. The cells were lysed using a sonicator
under cold conditions to preserve enzyme activity. Purification was conducted using nickel affinity chromatography,
with sequential washes using low-imidazole buffer A and high-imidazole buffer B. As shown in Figure 23, distinct
protein bands were observed in lysate samples. A ~39 kDa band was detected in the Cajanus-expressing strain,
consistent with the expected molecular weight of XCP2-Cicer. These results confirmed that recombinant enzymes were
successfully expressed in *E.coli*.

![XCP2-Cicer arietinum 蛋白图](https://static.igem.wiki/teams/5764/engineering/23.webp)

**Figure 23. SDS-PAGE result of pET28a-XCP2-Cicer**

**Learn:**

Similar to other isoenzymes, Cicer forms hydrogen
bond with substrates which leads to a high stability. However, it has less affinity caused by its different binding
sites. The prediction is proved by our ELISA test.

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

**1. Papain-mediated Casein Clot Dissolution
Assay**

To evaluate the initial proteolytic potential of the
recombinant enzyme papain, we conducted a qualitative assay based on its ability to degrade acid-precipitated casein
from milk.

As illustrated in Figure 25, untreated milk
containing 6% (w/v) casein appeared uniformly dispersed (A). The addition of dilute hydrochloric acid caused the
formation of a dense white precipitate, indicating the aggregation of denatured casein proteins (B). Following
incubation with papain at 37 °C for 1 hour, the precipitate visibly diminished and the solution became notably clearer
(C), indicating enzymatic breakdown of the protein network.

These results confirm that papain can hydrolyze
casein aggregates and exhibits functional caseinolytic activity. While this assay qualitatively supports its protease
function, it does not allow for comparison between different enzyme efficiencies.

![](https://static.igem.wiki/teams/5764/engineering/24.webp)

**Figure 24. Result of casein clot dissolution assay**

**2. FN1 Matrix Dissolution Assay**

To investigate whether the recombinant proteases are
capable of degrading biologically relevant substrates, fibronectin-1 (FN1) was chosen as the model protein for
enzymatic digestion.

As depicted in Figure 25, the FN1 control sample
(without enzyme treatment) showed a single, sharp band at approximately 47 kDa, indicating that the protein remained
intact in the absence of enzymatic activity. In contrast, samples treated with each of the five enzymes—Papain, Abrus,
Actinidia, Cajanus, and Cicer—exhibited a clear decrease in the intensity of the full-length FN1 band. Additionally,
prominent new bands emerged near 24 kDa, reflecting the formation of low-molecular-weight fragments resulting from
proteolytic cleavage.

The consistent generation of these ~24 kDa bands
across all enzyme groups suggests that, despite originating from different species, the recombinant proteases may
share similar substrate recognition or cleavage patterns. These findings demonstrate that all five enzymes are active
against FN1 and may be effective tools in applications related to extracellular matrix degradation or remodeling.

![](https://static.igem.wiki/teams/5764/engineering/25.webp)

**Figure 25. Result of FN1 matrix dissolution assay**

**3. ELISA-based Enzyme Activity Assay**

To measure and compare the proteolytic activity of
different crude enzyme lysates, we carried out an ELISA-based quantification assay. Prior to analysis, total protein
concentrations were determined using NanoDrop, and each lysate was diluted to various working concentrations. A
standard curve was constructed from serial dilutions of the kit’s reference standard, generating a linear model that
allowed us to estimate relative enzymatic activities from OD₄₅₀ values.

To quantitatively evaluate the enzymatic activity of
the crude lysates, an ELISA assay was performed. Protein concentrations were first normalized using NanoDrop, and all
lysates were diluted to different concentrations.

![标曲表格](https://static.igem.wiki/teams/5764/engineering/26.webp)

**Figure 26. The standard table of papain concentration**

A standard curve was generated based on serial
dilutions of the ELISA kit standard, yielding a linear regression model for activity estimation.

**![标曲1](https://static.igem.wiki/teams/5764/engineering/27.webp)**

**Figure 27. The standard curve of papain
concentration**

We used ELISA kits to individually test and analyze
the activity of several enzymes. Based on the above standard curves, we calculated the activity of each enzyme and
performed significance analysis using Graphpad to calculate the P-value. As shown in Figure 28, the enzyme activity
was *Papain > Act > Abr > Caj > Cic*.

![单酶ELISA表格](https://static.igem.wiki/teams/5764/engineering/28.webp)

**Figure 28. Single enzyme activity detection data**

Single-enzyme activity results showed that Papain had
the highest protease activity among all five enzymes, consistent with the qualitative results from the casein assay.
No statistically significant differences were observed between *Cajanus* and *Cicer*.

![单个酶活比较图](https://static.igem.wiki/teams/5764/engineering/29.webp)

**Figure 29. Graphs of single-enzyme activity comparison**

We used an ELISA kit to analyze the activity of the
dual-enzyme combinations and verify whether there was a synergistic effect. Based on the standard curve, we calculated
the activity of the dual-enzyme combinations and performed a significance analysis using Graphpad to calculate the
P-value. As shown in Figure 30, the optimal combination was *Caj+Cic*.

![双酶联用表格](https://static.igem.wiki/teams/5764/engineering/30.webp)

**Figure 30. Dual-enzyme activity detection data**

In the dual-enzyme combinations, the mixture of
*Cajanus* and *Cicer* displayed the highest activity. Other combinations, such as *papain* with
*Cajanus* or *Cicer,* and *Abrus* with *Actinidia*, showed moderate activity but no
significant enhancement compared to single enzymes.

![双酶联用比较](https://static.igem.wiki/teams/5764/engineering/31.webp)

**Figure 31. Graphs of dual-enzyme combination comparison**

We used an ELISA kit to analyze the activity of the
Three-enzyme combination and verify whether there was a synergistic effect. Based on the standard curve, we calculated
the activity of the Three-enzyme combination and performed a significance analysis using Graphpad to calculate the P
value. The optimal combinations were *Papain + Abrus + Actinidia* and *Abrus + Actinidia + Cajanus*.

![3酶联用表格](https://static.igem.wiki/teams/5764/engineering/32.webp)

**Figure 32. Three-enzyme activity detection data**

Three-enzyme combinations revealed that the groups
*Papain + Abrus + Actinidia* and *Abrus + Actinidia + Cajanus* produced the strongest activity levels,
although their differences were not statistically significant. These combinations outperformed most others in the same
group.

![三酶联用比较](https://static.igem.wiki/teams/5764/engineering/33.webp)

**Figure 33. Graphs of Three-enzyme combinations comparison**

We used an ELISA kit to analyze the activity of the
four-enzyme combination and verify whether there was a synergistic effect. Based on the standard curve, we calculated
the activity of the four-enzyme combination and performed a significance analysis using Graphpad to calculate the P
value. The optimal combination was *Abrus + Actinidia + Cajanus + Cicer*.

![D:/LYJ/2025-iGEM/2025-wiki/领科B-wiki/领科B wiki/8-Result/19.jpg19](https://static.igem.wiki/teams/5764/engineering/34.webp)

**Figure 34. Four-enzyme activity detection data**

In the four-enzyme combinations, the mixture of
*Abrus + Actinidia + Cajanus + Cicer* showed the highest value, indicating the strongest activity among all
tested combinations of four enzymes.

![四酶联用](https://static.igem.wiki/teams/5764/engineering/35.webp)

**Figure 35. Graphs of four-enzyme combinations comparison**

We used an ELISA kit to analyze the activity of the
five-enzyme combination and verify whether there was a synergistic effect. Based on the standard curve, we calculated
the activity of the five-enzyme combination and performed a significance analysis using Graphpad to calculate the P
value.Subsequently, we compared the combinations with the strongest enzyme activity under different combinations. The
results showed that the dual enzyme combination had the strongest effect.

![最佳协同比较表格](https://static.igem.wiki/teams/5764/engineering/36.webp)

**Figure 36. Different combinations activity detection data**

As shown in Figure 37, the dual combination of
*Cajanus + Cicer* displayed the highest overall activity, even exceeding that of the full five-enzyme mixture.
This suggests that specific pairings may exhibit synergistic effects stronger than broader mixtures.

![最优组合比较图](https://static.igem.wiki/teams/5764/engineering/37.webp)

**Figure 37. Graphs of optimal combinations comparison**

Among the five individual enzymes tested, Papain
demonstrated the strongest activity, in line with its rapid casein-clearing effect observed earlier. Cajanus and Cicer
showed similar performance, and no statistically significant difference was detected between them.

Interestingly, the Cajanus + Cicer combination
outperformed even the full five-enzyme mix, highlighting that targeted pairing of compatible enzymes can deliver
higher activity than complex multi-enzyme blends. This observation underscores the value of rational enzyme pairing in
optimizing proteolytic systems.

'
}]%}
{% block title %}Engineering{% endblock %}
{% block lead %}
![](https://static.igem.wiki/teams/5764/header/v1/engineering.webp)
{% endblock %}

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

**Goal:** To create a
functional and
nutritional liquid or solid environment for bacteria to grow. Liquid media used for various purposes such as
propagation of microorganism.

**Materials:** Sterilized
Duran
bottle\\Autoclave\\Tryptone\\Yeast Extract\\Sodium chloride\\Double distilled H2O\\Lab Refrigerator.

**Procedures for LB
solution:**

1. Prepare 20g of premix LB broth powder (Tryptone:Yeast extract:Sodium chloride-2:1:1).
2. Add into sterilized 1L bottle.
3. Add double distilled water to total volume of 1000ml.
4. Close the cap and autoclave it for 20 minutes at least.
5. Wait until it cools.

'
},
{
"title": "The construction of plasmid",
"content":'

1. **Obtaining target gene fragments by PCR**

**Goal:** To amplify
Papain\\Abrus\\Actinidia\\Cajanu\\Cicer fragments for later recombination.

**Materials:**2xTaq PCR Mix\*\\Papain\\Abrus\\Actinidia\\Cajanu\\Cicer gene
template\\ddH2O\\Primer-R\\Primer-F\\centrifuge tube\\ PCR thermal cycler.

\* 2xTaq PCR Mix includes
MgCl2, dNTP, Taq
DNA Polymerase, PCR buffer and loading buffer for PCR.

**Procedure:**

1. Add 2xTaq PCR Mix 25μl, papain template 2μl, primer-R 2μl, and primer-F 2μl to centrifuge tube. Add
   ddH2O to the centrifuge tube until reaching 50μl.
2. Centrifuge the mixture for a few seconds to remove the bubbles.
3. Insert mixture into a PCR thermal cycler.
4. Perform the cycle,Step2-4 repeat 30 cycles.

|  |  |  |
| --- | --- | --- |
| 94˚C | 5 minutes | Step 1 |
| 94˚C | 30 seconds | Step 2 |
| 55˚C | 30 seconds | Step 3 |
| 72˚C | 90 seconds | Step 4 |
| 72˚C | 10 minutes | Step 5 |

5. Repeat step 1-5 for *Abrus, Actinidia, Cajanus* and *Cicer.*

2. **Agarose gel construction, deployment and recycle**

**Goal:** To produce a
functional and
well-shaped gel for electrophoresis.

**Materials:** Casting
Tray\\Well
comb\\Microwave oven\\ 1x TAE\\Agarose\\10000x Nucleic acid gel stain\\Erlenmeyer flask.

**Procedure:**

1. Dissolve 0.5g Agarose in Erlenmeyer flask.
2. Add 50μL of TAE (Running buffer).
3. Heat the material for 3 minutes in microwave oven.
4. Add 3μl nucleic acid gel stain (10000x).
5. Let it cool for 10 minutes.
6. Pour the agarose mixture into the casting tray.
7. Place the appropriate well comb to create the wells.
8. Wait to solidify 15 minutes.
9. Remove the comb and place the gel in the gel box.

3. **Agarose gel electrophoresis**

**Goal:** To verify the
DNA bands\'
lengths and thus preparing for the next step of gel extraction to extract the bands that are of the correct length
(corresponding with the length of *Papain, Papain, Abrus, Actinidia, Cajanus* and *Cicer*),
eliminating
the genetic fragments that didn\'t successfully undergo PCR.

**Materials:** Marker
(DNA
ladder)\\Prepared gel\\10x Loading buffer\\Horizontal gel electrophoresis system.

**Procedure:**

1. Extract 10µl of every sample; add 5µl of loading buffer (10x) to each.
2. Place the gel in the horizontal electrophoresis system.
3. Extract 10µl of the mixed solution containing the sample, mix them till their colors are almost uniform.
4. Add the marker to the first well and load 10µl of each sample into the wells, the procedure runs at 180V for
   20min.
5. Afterwards, take out the gel from the device and proceed to following steps.

4. **DNA Gel Extraction**

**Goal:** To recover
DNA fragments
in agarose gel.

**Materials:**clean
scalpel\\ 1.5 mL
Eppendorf tube\\pipettes and sterilized pipette heads\\ hot water bath\\buffer B2\\purification column and
collection
tube\\centrifuge\\wash solution containing pure ethanol\\ddH2O.

**Procedure:**

1. Cut the slice of gel containing *Papain, Papain, Abrus, Actinidia, Cajanus* and *Cicer*, cutting
   off
   as much unneeded gel as possible, and place it in an 1.5 mL Eppendorf tube.
2. Add 500uL of buffer B2 and put the tube into a 50°C hot water bath until gel has completely melted.
3. Transfer the solution containing melted gel into a purification column that\'s in a collection tube and perform
   centrifugation at 8000 g for 30 seconds.
4. Empty the collection tube, put the purification column back in, and add 500uL of wash solution containing pure
   ethanol. Perform centrifugation at 9000 g for 30 seconds, and empty collection tube again.
5. Repeat (4).
6. Perform centrifugation one more time at 9000 g for 2 minute, then open the cap of the tube and let it sit for
   one
   minute to allow the ethanol to evaporate.
7. Transfer purification column to a 1.5 mL EP tube. Add 20uL of ddH2O at the center of the purification
   column.
8. Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 9000 g for 1 minute.
9. Discard the purification column. Store DNA at 4°C.

5. **Double digestion**

**Materials:** 1.5mL
Eppendorf
tube\\10×buffer\\NdeI\\HindⅢ\\pET28a\\ddH2O.

**Procedure:**

1. **Prepare the samples**

|  |  |
| --- | --- |
| Material | Amount |
| 10×buffer | 5µl |
| NdeI | 1µl |
| HindⅢ | 1µl |
| pET28a | 10µl |
| ddH2O | 33µl |

React in 37℃ for 30 minutes, Store
in 4℃.

6. **Homologous recombination**

**Goal:** add target
gene into vector so
that the gene could be transformed into competent cells for expression in the future.

**Material:** 2×CEⅡ
buffer\\Linear
pET28a\\pET28a-Papain/Abrus/Actinidia/Cajanus/Cicer

**Procedure:**

1. Prepare systems

|  |  |
| --- | --- |
| Material | Amount |
| 2×CEⅡ buffer | 5µl |
| Linear pET28a | 4µl |
| pET28a-papain\\... | 1µl |

React in 50℃ for 30 minutes,Store in
4℃.

7. **Extracted Plasmid**

**Materials:**Centrifuge\\1.5 ml EP
tube\\Collecting tube\\Buffer S\\Buffer sp1\\Buffer sp2\\Buffer sp3\\Buffer DW1\\Washing buffer 80%
ethanol\\ddH2O

**Procedure:**

1. Take out the cultured bacteria (if LB medium is opaque, then the bacteria has grown),fill both with 1.5ml of the
   bacteria-containing culture,centrifuge at 8,000xg for 2mins.
2. Add 250µl of buffer sp1, gently blow the bacterium using the micropipette to resuspend the bacteria.
3. Add 250µl of buffer sp2,Immediately gently mix the mixture for 5~10 times,incubate for 2~4 mins (no more than
   5min) at room temperature.
4. Add buffer sp3,Spin at a max of the centrifuge (greater or equal to 12,000xg) for 5~10 mins.
5. Carefully extract 700µl of the supernatant to the separating column (careful NOT to obtain any of the condensed
   DNA),Spin at 9,000xg for 30sec,Dispose of the liquid.
6. Add 500µl buffer DW1 to remove protein impurities,Spin at 9,000xg for 30sec,Dispose of the liquid.
7. Add 500µl wash solution and spin at 9,000xg for 30sec,Dispose of the liquid.
8. Repeat steps 7.
9. Spin at 9,000xg for 1min.
10. Use 20µl of 55℃ ddH2O and drop it at the very center of the membrane to wash off the plasmid.
11. Preserve the plasmids obtained at -20℃ for future usage.

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

1. **Culture**

**Goal:** to acquire
large amounts of
target proteins.

**Materials:**liquid
LB\\pET28a-Abrus/Actinidia/Cajanus/Cicer E-coli fluid\\Kanna antibiotic\\isothermic shaker.

**Procedure:**

1. Add 100μl of pET28a-Papain E-coli fluid and 100μl of kana into 100mL of liquid LB.
2. Place the sample in the isothermic shaker for one night in 37°C, 220rpm.
3. Change to other E-coli-fluids. Repeat the steps.

2. **Protein Extraction and SDS-PAGE**

**Goal:** to verify the
expression of
desired proteins.

**Materials:**bacteria
culture
containing desired protein\\50 mL centrifuge tubes\\beaker containing ice\\Centrifuge\\sonicator

**Procedure:**

1. add samples into 50 mL centrifuge tubes, perform centrifugation at 50% power for 10 minutes.
2. partially bury a centrifuge tube into a beaker filled with ice, then put the beaker and tube into the sonicator.
3. sonicate for 10 minutes in 50% power, turning on 3 seconds and stopping for 3 seconds for one circle.
4. repeat steps 2 and 3 for all samples.
5. the remaining liquid will contain desired proteins.

3. **SDS-PAGE:**

**Materials:**The SDS-PAGE color
preparation kit\\pipette\\distilled water\\electrophoresis buffer\\protein ladder\\vertical electrophoresis system

**Procedure：**

1. using a pipette, add and mix 4 mL of 2X separating gel solution, 4 mL of 2X separating gel buffer, and 80 μL of
   the catalyst in a plastic cup.
2. slowly inject mixture into casting stand and frame to avoid bubbles.
3. add 1 mL water to flatten out the top.wait 8 minutes for the gel to set.
4. discard the water that was added previously.
5. using a pipette, add and mix 1000 μL of 2X stacking gel solution, 1000 μL of 2X stacking gel buffer, and 20μL of
   catalyst in a plastic cup.
6. add stacking gel mixture until the cast is completely filled, then slowly insert comb without producing any air
   bubbles.
7. wait 12 minutes for the gel to set, carefully remove the comb, and wash the wells with electrophoresis buffer.
8. add the protein ladder into the first well, then load samples in each successive well.transfer gel into vertical
   electrophoresis system.
9. set the voltage to 180V and run it for 40 minutes.
10. stain the gel with Coomassie blue by submerging it for 10 minutes.

'},{
"title": "Functional verification",
"content":'

1. **Hydrolysis of casein**

**Goal:** Ensure the enzyme have the
ability to hydrolysis the casein

**Material:** milk\\The enzymes

**Procedure:**

1. Add acetic acid into the milk. Make the milk precipitate.
2. Add the enzyme
3. Measure the rate of casein hydrolysis

2. **Hydrolysis of FN1 protein**

**Goal:**
to ensure the enzyme have the ability to hydrolysis the FN1 protein

**Materials:** Papain samples\\Human
fibronectin (FN1)\\Activation buffer

**Procedures:**

1. Dissolve papain in the activation buffer. Let it stand at room temperature for 10 minutes to activate the enzyme
   activity.
2. Treatment of FN1 substrate dilute FN1 to 1 mg/mL with the activation buffer.
3. Add 10 μL of the activated enzyme solution to the FN1 substrate tube.
4. Immediately place it in a 37°C water bath for incubation for 2 hours after mixing.
5. Add 20 μL of SDS - PAGE loading buffer and heat at 95°C for 5 minutes. After precipitation on ice for 10
   minutes,
   centrifuge at 12,000 rpm for 10 minutes. Discard the supernatant and resuspend the precipitate in the buffer.
6. Load 20 μL of the terminated sample for SDS - PAGE detection.

3. **ELISA**

**Goal:**To test the efficiency when using different combinations of enzymes

**Material:**Enzyme detector
(450nm)\\37°C incubator\\double distilled water\\20×buffer\\substrate A\\substrate B\\stop buffer

**Procedure:**

1. Take the required strips from the aluminum foil bag that has been balanced at room temperature for 20 minutes.
   The
   remaining strips should be sealed in self-sealing bags and returned to 49°C.
2. Set up the standard sample wells and sample wells. Add 50 mL of samples of different concentrations to each
   standard sample well.
3. Add 50 mL of the sample to the sample wells; the blank wells do not receive any addition.
4. Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well
   of
   the standard sample wells and the sample wells. Seal the reaction wells with a sealing film and incubate at 37°C
   in
   a water bath or an incubator for 60 minutes.
5. Discard the liquid, blot dry with absorbent paper, and fill each well with washing solution (350 μL). Let it
   stand
   for 1 minute, then remove the washing solution and blot dry with absorbent paper. Repeat this process of washing
   the
   plates 5 times (or use a plate washer).
6. Add 50 μL of substrate A and B to each well. Incubate at 37°C in the dark for 15 minutes.
7. Add 50 μL of stop solution to each well. Measure the OD value of each well at 450 nm within 15 minutes.

'
}]%}
{% block title %}Experiments{% endblock %}
{% block lead %}
![](https://static.igem.wiki/teams/5764/header/v1/experiments.webp)
{% endblock %}

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

Our project aims to enhance skincare formulations
through the utilization of isoenzymes derived from papain, a cysteine protease renowned for its gentle exfoliating and
moisturizing properties. Papain functions by breaking down keratin proteins in the stratum corneum, thereby
facilitating the removal of dead skin cells and promoting skin regeneration. Compared to conventional acid-based
peels, papain offers a far gentler and more biocompatible alternative for exfoliation.

To develop superior enzymatic skincare agents, our
research focused on identifying papain isoenzymes that exhibit optimal activity across diverse skin types and pH
conditions. Using NCBI BLAST, genes with 50%-80% sequence similarity to papain were selected as candidate isoenzymes.
Structural modeling of these candidates was then performed via AlphaFold to evaluate and prioritize enzymes with
favorable active site characteristics. Ultimately, five cysteine proteases were chosen for their promising suitability
in skincare applications: those derived from *Abrus precatorius, Actinidia eriantha, Cicer arietinum, Cajanus
cajan*, and *papain* itself. These enzymes are expected to act synergistically to enhance the efficacy,
stability, and safety of enzymatic skincare products.

![流程图-01](https://static.igem.wiki/teams/5764/result/1.webp)

Figure 1. Overview of Experimental Steps

'
},{
"title": "Plasmid construction",
"content": '
**1.1 PCR Amplification**

The DNA sequence was amplified using PCR to produce
adequate quantities for downstream cloning in *E.coli* DH5α later on. The *E.coli* DH5α containing the
recombinant plasmids are also amplified. Figure 2 shows the agarose gel electrophoresis results of PCR for five clones.
We can find out that the DNA fragment of *Papain* is approximately 678 bp in length, *Abrus precatorius*is 1083 bp, *Actinidia eriantha* is 1089 bp, *Cajanus cajan* is 1083 bp and *Cicer
arietinum* is 1095 bp, which is consistent with the predicted size. This indicates that the target gene was
successfully amplified.

![PCR图_画板 1](https://static.igem.wiki/teams/5764/result/2.webp)

Figure 2. Agarose gel electrophoresis results of PCR for
five clones

**1.2 Homologous Recombination**

In this study, double digestion was employed to cut
pET28a plasmid vector with two restriction enzymes NdeI and HindIII, producing specific overhangs for the insertion of
the target gene. The gene fragment was designed with approximately 15-20 bp of homologous sequences at both ends,
matching the terminal regions of the linearized plasmid. These homologous overlaps facilitated precise insertion
through Exnase-mediated homologous recombination. The Exnase enzyme catalyzed the recombination between the plasmid
ends and the corresponding homologous sequences of the gene, resulting in seamless and directional integration without
the need for ligase.

After homologous recombination, the recombinant
plasmids were transformed into chemically competent *E. coli* DH5α cells via the heat shock method. Following
overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies were
observed (Figure 3), indicating successful transformation.

**![](https://static.igem.wiki/teams/5764/result/3.webp)**

Figure 3. Agar plate showing DH5α colonies after
transformation

To verify the presence of the target gene, five
individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 4.), each
colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion of
the target gene.

**![菌落鉴定_画板 1](https://static.igem.wiki/teams/5764/result/4.webp)**

Figure 4. Agarose gel electrophoresis results of Colony
PCR identification for five clones

**1.3 DNA Sequencing**

To confirm the accuracy of plasmid construction,
recombinant pET28a plasmids from five positive clones were sent to a commercial sequencing facility for Sanger
sequencing. Sequence alignment (Figure 5.) showed that the inserted gene fragments matched the designed sequences
exactly, with no frameshift mutations or base substitutions, indicating successful and accurate cloning of the target
genes.

**![测序图_画板 1](https://static.igem.wiki/teams/5764/result/5.webp)**

Figure 5. DNA sequencing results of Recombinant plasmid

'
}, {
"title": "Protein expression",
"content": '
**2.1 Heat shock conversion into BL21(DE3)**

Each of the five recombinant plasmids encoding
papain, Abrus, Actinidia, Cajanus, and Cicer proteases was independently transformed into *E. coli* BL21(DE3)
competent cells using the heat shock method. The competent cells were first incubated with plasmid DNA on ice and then
exposed to a brief heat shock at 42 °C to facilitate DNA uptake. Following recovery in LB medium, the cells were
plated on kanamycin-selective LB agar plates and incubated overnight at 37 °C.

As shown in Figure 6, well-isolated single colonies
appeared for each transformation, indicating successful uptake of the recombinant plasmids. One colony from each
transformation was randomly selected for colony PCR. The agarose gel electrophoresis results confirmed the presence of
the target genes, verifying successful transformation and plasmid integrity in all five constructs.

**![](https://static.igem.wiki/teams/5764/result/6.webp)**

Figure 6. Agar plate showing BL21 colonies after
transformation

**2.2 Lysate protein concentration**

Each PCR-verified colony was inoculated into liquid
LB medium and cultured overnight, followed by scale-up fermentation. After cell harvest via centrifugation, bacterial
pellets were subjected to ultrasonic lysis to release intracellular contents. Crude protein was extracted from the
lysates without further purification at this stage.

Protein concentration was determined for each lysate
using the Bradford assay. As shown in Figure 7, the total protein concentrations varied slightly among strains but were
all within an acceptable range for downstream purification and activity assays.

![粗蛋白浓度表格](https://static.igem.wiki/teams/5764/result/7.webp)

Figure 7. Concentration of five lysate protein

**2.3 SDS-PAGE verification**

To verify the expression of recombinant proteins and
evaluate their approximate molecular weights, SDS-PAGE analysis was performed on both crude and purified samples.
Proteins were separated on a 15% polyacrylamide gel and stained with Coomassie Brilliant Blue.

As shown in Figure 8, distinct protein bands were
observed in all five lysate samples. A ~24 kDa band was detected in the papain-expressing strain, consistent with the
expected molecular weight of papain. For *Abrus*, *Actinidia*, *Cajanus*, and *Cicer*
proteases, bands were observed around ~39 kDa, matching their predicted sizes. These results confirmed that all five
recombinant enzymes were successfully expressed in *E. coli*.

**![蛋白图_画板 1](https://static.igem.wiki/teams/5764/result/8.webp)**

Figure 8. SDS-PAGE Analysis Before and After Protein
Elution

'
}, {
"title": "Activity Validation",
"content": '**3.1 Casein Clot dissolution Assay**

To preliminarily assess proteolytic activity, the
recombinant enzyme papain was tested for its ability to dissolve acid-induced casein precipitates in milk.

As shown in Figure 9, fresh milk containing 6% (w/v)
casein appeared as a uniform liquid (Figure 9A). Upon mixing with dilute hydrochloric acid, a visible white precipitate
formed, representing denatured casein aggregates (Figure 9B). After treatment with papain for 1 hour at 37 °C, the
precipitate significantly reduced, and the solution became clearer (Figure 9C), suggesting successful hydrolysis of the
protein clot.

This observation demonstrates that papain possesses
caseinolytic activity and can cleave aggregated casein chains. The experiment provides qualitative evidence of
protease activity but does not differentiate between the relative activity levels of different enzymes.

**![水解酪蛋白实验图](https://static.igem.wiki/teams/5764/result/9.webp)**

Figure 9. Results of casein hydrolysis

**3.2 FN1 Matrix Dissolution Assay**

To evaluate whether the recombinant enzymes possess
proteolytic activity toward physiologically relevant substrates, fibronectin-1 (FN1) was selected for hydrolysis
assays.

As shown in Figure 10, the FN1 protein appeared as a
single intact band at approximately 47 kDa in the blank group, indicating structural stability in the absence of
active enzymes. In contrast, all five enzyme-treated samples *(Papain*, *Abrus*, *Actinidia*,
*Cajanus*, and *Cicer*) displayed a significant reduction in the 47 kDa FN1 band intensity.
Simultaneously, distinct new bands appeared around 24 kDa, suggesting enzymatic cleavage of FN1 into smaller peptide
fragments.

The consistent presence of ~24 kDa fragments across
all treatment groups implies that the enzymes—despite their different sources—may hydrolyze FN1 at similar cleavage
sites. This result confirms that all five recombinant proteases exhibit biological activity against FN1, supporting
their potential for applications involving ECM protein remodeling.

**![木瓜酶水解1-01](https://static.igem.wiki/teams/5764/result/10.webp)**

Figure 10. Results of FN1 proteolytic digestion

**3.3 ELISA-based Enzyme Activity Assay**

To quantitatively evaluate the enzymatic activity of
the crude lysates, an ELISA assay was performed. Protein concentrations were first normalized using NanoDrop, and all
lysates were diluted to different concentrations.

![标曲表格](https://static.igem.wiki/teams/5764/result/11.webp)

Figure 11. The standard table of papain concentration

A standard curve was generated based on serial
dilutions of the ELISA kit standard (Figure 12), yielding a linear regression model for activity estimation.

**![标曲1](https://static.igem.wiki/teams/5764/result/12.webp)**

Figure 12. The standard curve of papain concentration

We used ELISA kits to individually test and analyze
the activity of several enzymes. Based on the above standard curves, we calculated the activity of each enzyme and
performed significance analysis using Graphpad to calculate the P-value. As shown in Figureure 13, the enzyme activity
was *Papain > Act > Abr > Caj > Cic*.

![单酶ELISA表格](https://static.igem.wiki/teams/5764/result/13.webp)

Figure 13. Single enzyme activity detection data

Single-enzyme activity results (Figure 14) showed that
Papain had the highest protease activity among all five enzymes, consistent with the qualitative results from the
casein assay. No statistically significant differences were observed between *Cajanus* and *Cicer*.

![单个酶活比较图](https://static.igem.wiki/teams/5764/result/14.webp)

Figure 14. Graphs of single-enzyme activity comparison

We used an ELISA kit to analyze the activity of the
dual-enzyme combinations and verify whether there was a synergistic effect. Based on the standard curve, we calculated
the activity of the dual-enzyme combinations and performed a significance analysis using Graphpad to calculate the
P-value. As shown in Figure 15, the optimal combination was *Caj+Cic*.

![双酶联用表格](https://static.igem.wiki/teams/5764/result/15.webp)

Figure 15. Dual-enzyme activity detection data

In the dual-enzyme combinations (Figure 16), the mixture
of *Cajanus* and *Cicer* displayed the highest activity. Other combinations, such as *papain*with *Cajanus* or *Cicer,* and *Abrus* with *Actinidia*, showed moderate activity but
no significant enhancement compared to single enzymes.

![双酶联用比较](https://static.igem.wiki/teams/5764/result/16.webp)

Figure 16. Graphs of dual-enzyme combination comparison

We used an ELISA kit to analyze the activity of the
Three-enzyme combination and verify whether there was a synergistic effect. Based on the standard curve, we calculated
the activity of the Three-enzyme combination and performed a significance analysis using Graphpad to calculate the P
value. As shown in Figure 17, the optimal combinations were *Papain + Abrus + Actinidia* and *Abrus + Actinidia
+ Cajanus*.

![3酶联用表格](https://static.igem.wiki/teams/5764/result/17.webp)

Figure 17. Three-enzyme activity detection data

Three-enzyme combinations (Figure 18) revealed that the
groups *Papain + Abrus + Actinidia* and *Abrus + Actinidia + Cajanus* produced the strongest activity
levels, although their differences were not statistically significant. These combinations outperformed most others in
the same group.

![三酶联用比较](https://static.igem.wiki/teams/5764/result/18.webp)

Figure 18. Graphs of Three-enzyme combinations
comparison

We used an ELISA kit to analyze the activity of the
four-enzyme combination and verify whether there was a synergistic effect. Based on the standard curve, we calculated
the activity of the four-enzyme combination and performed a significance analysis using Graphpad to calculate the P
value. As shown in Figure 19, the optimal combination was *Abrus + Actinidia + Cajanus + Cicer*.

![4酶联用表格](https://static.igem.wiki/teams/5764/result/19.webp)

Figure 19. Four-enzyme activity detection data

In the four-enzyme combinations (Figure 20), the mixture
of *Abrus + Actinidia + Cajanus + Cicer* showed the highest value, indicating the strongest activity among all
tested combinations of four enzymes.

![四酶联用](https://static.igem.wiki/teams/5764/result/20.webp)

Figure 20. Graphs of four-enzyme combinations comparison

We used an ELISA kit to analyze the activity of the
five-enzyme combination and verify whether there was a synergistic effect. Based on the standard curve, we calculated
the activity of the five-enzyme combination and performed a significance analysis using Graphpad to calculate the P
value, as shown in Figure 21.Subsequently, we compared the combinations with the strongest enzyme activity under
different combinations. The results showed that the dual enzyme combination had the strongest effect.

![最佳协同比较表格](https://static.igem.wiki/teams/5764/result/21.webp)

Figure 21. Different combinations activity detection
data

As shown in Figure 22, the dual combination of
*Cajanus + Cicer* displayed the highest overall activity, even exceeding that of the full five-enzyme mixture.
This suggests that specific pairings may exhibit synergistic effects stronger than broader mixtures.

![最优组合比较图](https://static.igem.wiki/teams/5764/result/22.webp)

Figure 22. Graphs of optimal combinations comparison

'
},{
"title": "Structural prediction",
"content": '
**4.1 NCBI Blast**

To explore structural similarities between papain and
other potential isoenzymes, a NCBI BLAST search was performed using the amino acid sequence of *Carica papaya
papain* as the query. As shown in Figure 23, we identified 30 homologous sequences with more than 50% sequence
identity and less than 80%.

![A table of information with numbers and letters  AI-generated content may be incorrect.](https://static.igem.wiki/teams/5764/result/23.webp)

Figure 23. Results of NCBI BLAST analysis

The amino acid sequences of these homologous enzymes
were successfully downloaded (Figure 24) and subsequently subjected to three-dimensional structure prediction using
AlphaFold2. The predicted structures provide a solid foundation for downstream molecular docking simulations, enabling
a structure-based analysis of binding behavior and functional conservation among papain-like proteases.

![图1](https://static.igem.wiki/teams/5764/result/24.webp)

Figure 24. Results of sequence alignment analysis

**4.2 Molecular Docking Simulation**

To evaluate binding potential, we conducted molecular
docking simulations between the predicted structures and the synthetic tripeptide substrate Pglu-Phe-Leu-pNA, commonly
used in protease activity assays.

As shown in Figure 25, Papain exhibited the strongest
binding affinity, as indicated by the lowest docking energy score. In addition, isoenzymes from *Actinidia
eriantha*, *Abrus precatorius*, *Cicer arietinum*, and *Cajanus cajan* also showed
relatively strong binding scores, suggesting their ability to form stable enzyme-substrate complexes. These findings
imply that the candidate enzymes may possess similar substrate recognition and catalytic properties as
*papain*.

![](https://static.igem.wiki/teams/5764/result/25.webp)

Figure 25. Indication of the binding affinity between
five enzymes and the substrate

**4.3 Binding Affinity Analysis**

Detailed analysis of the docked complexes revealed
that all five high-affinity enzymes formed dense hydrogen bonding networks with the substrate (Figure 26). Notably,
papain formed both hydrogen bonds and additional π–π stacking interactions, a unique feature not observed in the other
isoenzymes.

These π–π interactions are likely to contribute to
enhanced binding stability, potentially explaining why papain displayed the strongest affinity in docking simulations.
While the other isoenzymes also formed stable complexes, their lack of π–π interactions and subtle differences in
binding pocket geometry likely account for the variation in binding strength.

This binding affinity pattern is consistent with the
enzyme activity results obtained from ELISA assays, further validating the structural-functional correlation.
Together, the docking and binding affinity analyses strongly support the conclusion that microbially synthesized
papain retains functional features of plant-derived papain, laying the foundation for future application-oriented
studies.

**![图2](https://static.igem.wiki/teams/5764/result/26.webp)**

Figure 26. Results of molecular interaction analysis
between five enzymes and the substrate

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

These results not only confirm the proteolytic
activity and substrate-binding potential of the recombinant papain and its isoenzymes, but also provide key structural
and functional data as raw material for further studies on multi-enzyme synergy. For example, whether the enzymes act
simultaneously or sequentially during substrate degradation remains unknown and could be clarified through substrate
competition assays or reaction time-course studies.

Furthermore, the demonstrated activity of microbially
synthesized papain lays the foundation for its potential large-scale replacement of plant-derived papain, particularly
through alternative microbial hosts such as Bacillus subtilis or yeast, which may offer higher yields and better
post-translational modifications than *E. coli*.

To ensure applicability in biomedical or cosmetic
fields, toxicity, allergenicity, and safety assessments of the recombinant enzymes are also warranted. Collectively,
the current findings provide experimental and computational groundwork for optimizing enzyme formulation, improving
production strategies, and evaluating biosafety for future industrial applications.

'
}]%}
{% block title %}Results{% endblock %}
{% block lead %}
![](https://static.igem.wiki/teams/5764/header/v1/results.webp)
{% endblock %}