iGEM 2025 — Team njtech-china-a


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## Engineering

* [Introduction](#introduction)
* [Cecropin B](#cecropin-b)
* [Anionic Antioxidant Peptides](#anionic-antioxidant)
* [Signal Peptide](#signal-peptide)
* [Fermentation Optimize](#fermentation)
* [Mutation](#mutation)
* [Discovering of New Peptide](#new-peptide)
* [Molecular Chaperones](#molecular-chaperones)
* [Antibacterial Hydrogels](#antibacterial-hydrogels)
* [Optimization of Antibacterial Hydrogels](#hydrogels-optimization)
* [Outlook](#outlook)

## Introduction

Cecropin B (CecB) was originally isolated from the silkworm moth Hyalophora cecropia. It is a heat-stable and soluble peptide that exhibits broad-spectrum antimicrobial activity. CecB is effective against both Gram-positive and Gram-negative bacteria, as well as certain viruses, while showing minimal cytotoxicity toward eukaryotic cells. Due to its strong antimicrobial properties, the expression and purification of CecB are of great significance in antimicrobial peptide research. Currently, Escherichia coli is the most common host used for CecB expression. However, since CecB is also highly active against E. coli, its strong antimicrobial effect presents a key challenge for large-scale expression in this host. The interaction between positively charged AMPs and negatively charged bacterial membranes triggers the formation of cracks in the membranes, leading to pore formation and ultimately bacterial death. In addition to pore-forming AMPs, some AMPs can translocate across bacterial membrane barriers through self-promoted uptake and target key cellular processes, such as macromolecular synthesis (DNA, RNA, proteins, and cell walls), protein folding, and enzyme activity (Fig. 1).

![Mechanism of antimicrobial peptide action](https://static.igem.wiki/teams/5809/engineering/engineering-1.webp)

Fig. 1 Mechanism of antimicrobial peptide action[1]

We analyzed the sequence by performing a BLASTp search in the NCBI non-redundant protein sequence database. Codon optimization was carried out after removing non-clustered sequences. The resulting sequences were then subjected to structural modeling using AlphaFold2 (Fig. 2).

![The structure prediction diagram of cecropin B](https://static.igem.wiki/teams/5809/engineering/engineering-2.webp)

Fig. 2 The structure prediction diagram of cecropin B

## Cecropin B

### Design:

Through the iGEM Registry, we selected CecB as our antimicrobial peptide sequence. We performed a BLASTp search in the NCBI database using the protein sequence obtained from the Registry (Fig. 3), and carried out codon optimization with commercial codon optimization software. Primers were synthesized by a company, and we assembled the cecB fragment ourselves before inserting it into the pET-28a vector for subsequent expression in bacteria.

![Results of NCBI sequence alignment](https://static.igem.wiki/teams/5809/engineering/engineering-3.webp)

Fig. 3 Results of NCBI sequence alignment

The plasmid map and signal pathway are illustrated as follows:

![Map of the recombinant plasmid pET-28a-cecB](https://static.igem.wiki/teams/5809/engineering/engineering-4.webp)

Fig. 4 Map of the recombinant plasmid pET-28a-cecB

![Map of the recombinant plasmid pET-28a-cecB](https://static.igem.wiki/teams/5809/engineering/engineer-5.webp)

Fig. 5 CecB signaling pathway diagram

### Construction:

All primers were added into the PCR system for template-free amplification to synthesize the CecB fragment. The correct band was identified by AGE(agarose gel electrophoresis) (Fig. 6), recovered from the gel, and the plasmid was extracted.

The synthesized gene was then cloned into the plasmid and subsequently introduced into E. coli BL21 (DE3).

Transformed single colony were picked from the plates and cultured, followed by colony PCR verification to confirm the presence of the recombinant plasmid.

![Colony PCR results of cecB](https://static.igem.wiki/teams/5809/engineering/engineering-6.webp)

Fig. 6 Colony PCR results of cecB

![Sequencing chromatogram of cecB](https://static.igem.wiki/teams/5809/engineering/engineering-7.webp)

Fig. 7 Sequencing chromatogram of cecB

The correctly verified colonies (Fig. 7) were inoculated into 50 mL of LB medium for induced expression. After induction, the cells were lysed, and the supernatant was collected for purification and ultrafiltration. The total protein, ultrafiltration product, and pellet were separately analyzed by Western blot, and their antibacterial activity was evaluated through inhibition zone assays (Fig. 9).

### Test:

The molecular weight of CecB-EDPNG-6×His is 5.4 kDa (Fig. 8). The purified ultrafiltration product was used for inhibition zone (Fig. 9) and MIC assays.

![Western blot image of CecB-EDPNG-6Ã—His](https://static.igem.wiki/teams/5809/engineering/engineering-8.webp)

Fig. 8 Western blot image of CecB-EDPNG-6×His

Verification showed that no distinct protein bands were observed in the total protein, pellet, or ultrafiltration fractions.

![Results of inhibition zone assay](https://static.igem.wiki/teams/5809/engineering/engineering-9.webp)

Fig. 9 Results of inhibition zone assay

**1：PelB-Yin-CecB；2：Yin-CecB-1；3：CecB；4：Yin-CecB-2**

### Results:

Our measurements showed that no target band was detected in the Western blot. Furthermore, CecB exhibited no significant antibacterial effect in either inhibition zone assays or MIC tests.

### Learning:

From the inhibition zone experiment, we observed that CecB expression and antibacterial activity in E. coli were both unsatisfactory. We suspect that this may be due to the intrinsic antibacterial activity of Cecropin B against Gram-negative bacteria, which prevents it from being efficiently expressed in a suitable environment.

## Anionic Antioxidant Peptides

### Introduction:

We hypothesized that the positive charge of CecB exerts an inhibitory effect on E. coli and is toxic to its host. To address this, we introduced an anionic antioxidant peptide. Antioxidants can inhibit the formation of reactive oxygen species (ROS) that cause cellular or tissue damage. Synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) are restricted due to their potential risks. In this study, we used an anionic antioxidant peptide to neutralize the positive charge of CecB. Specifically, we selected an anionic antioxidant peptide derived from porcine myofibrillar protein the host. However, before application, the peptide must be cleaved. For this purpose, we employed EDPNG as a cleavage method, which is faster and more efficient compared to conventional approaches such as TEV protease or enterokinase.

### Design:

The anionic antioxidant peptide was inserted into the previously constructed pET-28a-cecB vector. The plasmid map is shown below (Fig. 10).

![pET-28a-yin-CecB](https://static.igem.wiki/teams/5809/engineering/engineering-10.webp)

Fig. 10 pET-28a-yin-CecB

![pET-28a-yin-CecB](https://static.igem.wiki/teams/5809/engineering/engineer-11.webp)

Fig. 11 Schematic diagram of the yin-cecB signaling pathway

The anionic antioxidant peptide and pET-28a were separately amplified from templates by PCR, and the correct PCR bands were recovered from agarose gel electrophoresis.

The recovered plasmid and insert were then adjusted to optimal concentrations and subjected to homologous recombination.

The recombinant plasmid was transformed into E. coli DH5α. After single colonies appeared, colony PCR was performed using T7 primers. The results are shown in the figure below (Fig. 12).

![yin-cecB colony PCR image](https://static.igem.wiki/teams/5809/engineering/engineering-12.webp)

Fig. 12 yin-cecB colony PCR image

The correctly verified colonies were sent for sequencing. Successfully sequenced clones were used for plasmid extraction and subsequently transformed into E. coli BL21 (DE3) for activation and induced expression.

![Sequencing chromatogram of yin-cecB](https://static.igem.wiki/teams/5809/engineering/engineering-13.webp)

Fig. 13 Sequencing chromatogram of yin-cecB

### Test:

One batch of the induced cultures was lysed and purified. Lysis buffer was then added to the purified sample to induce EDPNG cleavage. After a 4-hour reaction, the pH was adjusted to terminate the reaction, followed by ultrafiltration. Another batch was lysed, centrifuged, and lysis buffer was added directly to the supernatant, which was then lyophilized. The resulting samples were subjected to MIC and inhibition zone assays (Fig. 14).

![Inhibition assay results of Yin-CecB](https://static.igem.wiki/teams/5809/engineering/engineering-14.webp)

Fig. 14 Inhibition assay results of Yin-CecB

1：PelB-Yin-CecB；2：Yin-CecB-1；3：CecB；4：Yin-CecB-2

![MIC results of Yin-CecB](https://static.igem.wiki/teams/5809/engineering/engineering-15.webp)

Fig. 15 MIC results of Yin-CecB

### Results:

We found that after adding the anionic antioxidant peptide, a slight antibacterial effect was observed. The surrounding bacterial growth was noticeably reduced, and MIC results showed inhibition up to the second well. Notably, the lyophilized samples exhibited stronger antibacterial activity compared to those subjected directly to ultrafiltration.

### Learning:

We discovered that the addition of the anionic antioxidant peptide enhanced the antibacterial effect, extending inhibition to the second well. This indicates that our protective peptide indeed played a beneficial role.

## Signal Peptide

### Introduction:

After the anionic antioxidant peptide was linked to CecB, the total protein level increased. However, the E. coli expression system contains many host proteins that readily drive recombinant products into inclusion bodies or misfolded conformations. Therefore, we superimposed a signal peptide. The signal peptide can assist the protein in completing translocation and regulate the speed of protein translation, giving it more opportunities to fold correctly and be processed into an active protein. The so-called secretory expression in E. coli usually refers to the protein being translocated into the periplasm, the space between the outer and inner membranes, and then further translocated into the culture medium outside the cell membranetypically a two-step secretion. If the second step cannot be achieved, the protein can only remain in the periplasm. For most proteins, translocation to the periplasm allows for better disulfide bond oxidation and protein folding, resulting in relatively high activity. Proteins that are not directly secreted into the culture medium are also easier to collect and purify.

When exogenous recombinant proteins are expressed in E. coli, they often readily form inclusion bodies; the main reason is that recombinant protein expression occurs in the cytoplasm, the cytoplasm is a strongly reducing environment that is unfavorable for disulfide-bond formation and lacks the enzymes needed for disulfide-bond generation, whereas the periplasmic environment is very suitable for disulfide-bond formation and correct folding of recombinant proteins (disulfide-bond oxidoreductases and peptidyl-prolyl isomerases), and the periplasmic space is small, can store few proteins, and allows proteins to be slowly processed and correctly folded there.

### Design:

We completely integrated the PelB signal peptide into the pET-28a(+) plasmid vector.

![Signal pathway map of PelB-Yin-CecB](https://static.igem.wiki/teams/5809/engineering/engineering-16.webp)

Fig. 16 Plasmid map of pET-28a-pelB-yin-cecB

![Signal pathway map of PelB-Yin-CecB](https://static.igem.wiki/teams/5809/engineering/engineering-17.webp)

Fig. 17 Signal pathway map of PelB-Yin-CecB

### Construction:

1. The signal-peptide template and the pre-assembled fragment were each amplified by PCR. After agarose-gel electrophoresis, the correct bands were excised and the fragments were gel-purified; the plasmids were then extracted.
2. The purified fusion fragment was assembled with the vector by homologous recombination, and the mixture was heat-shock transformed into E. coli BL21(DE3).
3. Single colonies that grew on the plate were picked and cultured, and colony PCR was performed to verify the presence of the recombinant plasmid.

![Colony PCR of pelB-yin-cecB](https://static.igem.wiki/teams/5809/engineering/engineering-18.webp)

Fig. 18 Colony PCR of pelB-yin-cecB

Bacteria verified by colony PCR were sent for testing; bacteria whose sequencing was correct were cultured for induction expression. After induction expression, the supernatant obtained after cell disruption was purified, and twice the volume of lysis buffer was added to the purified product; the lysis reaction was carried out for 4h, then the pH was adjusted to stop the reaction, and the material after reaction was collected to do WB experiment (Fig. 19).

### Testing:

![PelB-Yin-CecB WB image](https://static.igem.wiki/teams/5809/engineering/engineering-19.webp)

Fig. 19 PelB-Yin-CecB WB image

**Far left is 10--180 kDa marker, lane 1 is ultrafiltered protein sample, lane 2 is total protein sample, lane 3 is protein in pellet (pellet resuspended with 5 mL PBS pH 7.4).**

Verification showed that the PelB-Yin-CecB protein was successfully expressed, and its content was significantly higher than the expression level of CecB alone. Since the effect of Yin-CecB after direct freeze-drying was better than that after purification and ultrafiltration, the disrupted protein was mixed with lysis buffer at a volume twice that of the protein to cleave the anionic antioxidant peptide. After freeze-drying, the CecB was obtained and used for MIC determination (Fig. 20) and antibacterial effect testing (Fig. 21).

![PelB-Yin-CecB MIC](https://static.igem.wiki/teams/5809/engineering/engineering-20.webp)

Fig. 20 PelB-Yin-CecB MIC

![Yin-CecB antibacterial effect image](https://static.igem.wiki/teams/5809/engineering/engineering-21.webp)

Fig. 21 Yin-CecB antibacterial effect image

**1：PelB-Yin-CecB；2：Yin-CecB-1；3：CecB；4：Yin-CecB-2**

### Results:

The overall OD values of Group 3 were relatively low, yet some trends still appeared, and the same trends were also observed in Group 2 and Group 3. We found that the antibacterial MIC reached the third well, and a clear protein band was present in the WB; the antibacterial effect was noticeably stronger than that of both Yin-CecB and CecB.

### Learning:

Comparison showed that after adding the signal peptide PelB the antibacterial activity was indeed higher than the other two, and the protein expression level increased, with most of the protein remaining in the supernatant rather than in the pellet. Although the liquid after purification and ultrafiltration gave high purity and good expression after freeze-drying, the process wasted too much time and medium; once this approach was omitted, no obvious antibacterial effect was seen, so we also optimized the fermentation conditions.

## Fermentation Optimize

### Design:

Fermentation temperature is one of the key factors affecting the E. coli expression system; excessively high induction temperatures prevent proper protein folding and lead to inclusion-body formation, trapping the protein in the pellet and preventing its release.

### Construction:

Because secretion through the periplasmic space is relatively slow, we investigated fermentation temperatures for the above PelB-Yin-CecB protein use 20h. Cultures were grown in LB medium, and fermentation broths at 16°C,25°C,30°C and 37°C were collected, disrupted and purified; protein concentrations were measured and expression was verified by SDS-PAGE.

As IPTG concentration during induction is also an important determinant of expression, we performed induction at final concentrations of 0.25 mM, 0.5 mM and 1 mM; the resulting broths were disrupted and purified, and the obtained protein samples were analyzed by SDS-PAGE.

To examine the effect of medium on CecB, we carried out a comparative experiment using both TB and LB media.

### Test:

Given that folding in the periplasmic space may be slow, we measured the protein content of fermentation products obtained after 20h of culture at the different temperatures.

![Optical density image of temperature](https://static.igem.wiki/teams/5809/engineering/engineering-22.webp)

Fig. 22 Optical density image of temperature

![Optical density image of temperature percentage](https://static.igem.wiki/teams/5809/engineering/engineering-23.webp)

Fig. 23 Optical density image of temperature

![Optical density image of IPTG Concentration](https://static.igem.wiki/teams/5809/engineering/engineering-24.webp)

Fig. 24 Optical density image of IPTG Concentration

![Optical density image of IPTG Concentration percentage](https://static.igem.wiki/teams/5809/engineering/engineering-25.webp)

Fig. 25 Optical density image of IPTG Concentration

![Optical density image of media optimization](https://static.igem.wiki/teams/5809/engineering/engineering-26.webp)

Fig. 26 Optical density image of media optimization

![Optical density image of media optimization percentage](https://static.igem.wiki/teams/5809/engineering/engineering-27.webp)

Fig. 27 Optical density image of media optimization

### Results:

As shown by the optical-density image, more bands were obtained in the supernatant when the temperature was around 16°C, and the percentage image further revealed that the protein content in the supernatant was highest; therefore, all subsequent inductions were performed at 16°C.

The IPTG-concentration optical-density image showed that, when the culture reached OD600 = 0.6, IPTG was added to a final concentration of 0.5 mM.

Medium optimization demonstrated that CecB carrying PelB was already barely detectable in the pellet, and after switching to TB medium the PelB-equipped protein appeared exclusively in the supernatant. For plasmids lacking the signal peptide, the amount of protein in the TB-medium supernatant also increased. However, without ultrafiltration followed by freeze-drying no obvious antibacterial effect was observed, a procedure that costs extra time and money and does not match our expectations.

### Learning:

In synthetic biology, choosing an optimal system requires consideration of several factors; optimizing the fermentation setup laid the foundation for improved CecB expression. Nevertheless, the observed protein yield and inhibition-zone size were far below expectations, so we next asked whether mutation might bring improvement.

## Mutation

### Introduction:

A pre-trained large language model was fine-tuned for a specific task based on the amino acid sequence of Cecropin B (CecB). The fine-tuning dataset comprised known antimicrobial peptide (AMP) sequences and their functional annotations, aiming to enhance the model's ability to identify key physicochemical features, including charge distribution, amphipathicity, and secondary structure propensity. The fine-tuned model generated candidate sequences featuring primarily single-point mutations while preserving the overall peptide backbone.

**Charge-enhanced variants:** Substitution with positively charged residues (e.g., Lys/Arg) to improve interaction with negatively charged targets.

**Structure-optimized variants:** Replacement with hydrophobic or small-volume residues to improve folding and binding groove compatibility.

All candidate sequences were systematically organized into a table for subsequent computational validation.

### Design:

Predictive modeling yielded the following mutagenesis strategies.

Table 1. Single mutation sequence

| Number | Name | Sequence |
| --- | --- | --- |
| M1 | E10K | MKWKVFKKIKKMGRNIRNGIVKAGPAIAVLGEAKALG |
| M2 | L37insR | MKWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKALRG |
| M3 | N15K | MKWKVFKKIEKMGRKIRNGIVKAGPAIAVLGEAKALG |
| M4 | P25A | MKWKVFKKIEKMGRNIRNGIVKAGAAIAVLGEAKALG |
| M5 | V29W | MKWKVFKKIEKMGRNIRNGIVKAGPAIAWLGEAKALG |
| M6 | M6 | MKWKVFKKIEKWWKKAGKWLKK |
| M7 | M7 | MKWKVFKKIEKMGRRKKIRWIKK |

Since M2, along with M1 and M3, were all designed for charge enhancement, we selected M1 and M3. M4 was chosen for structural optimization, and M5 for hydrophobicity optimization. M6 and M7 were excluded due to their extensive truncations. Consequently, the M1, M3, M4, and M5 mutations were ultimately selected for further investigation.

Accordingly, we designed mutagenesis primers targeting the pelB-cecB construct.

![Mutagenesis Primer Design Scheme](https://static.igem.wiki/teams/5809/engineering/engineering-29.webp)

Fig. 28 Mutagenesis Primer Design Scheme

### Construction:

Mutation sites were introduced into the pET-28a-pelB-yin-cecB plasmid using the designed primers. DNA fragments of the correct size, as verified by agarose gel electrophoresis, were purified using the FastPure Gel DNA Extraction Mini Kit (Vazyme, Nanjing, China). The parental plasmid template was digested with Dpn I, and circularized plasmids were obtained via a one-step cloning method, followed by heat-shock transformation into E. coli BL21(DE3) competent cells.

![Mutant Colony PCR Bands](https://static.igem.wiki/teams/5809/engineering/engineering-30.webp)

Fig. 29 Mutant Colony PCR Bands

**Lanes1&2:** T7-pelB-yin-cecB-E10K-T7t; **Lanes3&4:** T7-pelB-yin-cecB-N15K-T7t; **Lanes5&6:** T7-pelB-yin-cecB-P25A-T7t; **Lanes7&8:** T7-pelB-yin-cecB-V29W-T7t.

Colony PCR-positive clones were sent for commercial sequencing.

![Mutant Sequencing Results](https://static.igem.wiki/teams/5809/engineering/engineering-31.webp)

Fig. 30 Mutant Sequencing Results

Sequencing validation confirmed the successful incorporation of the E10K, N15K, and V29W mutations. In contrast, the P25A mutation was not obtained successfully, despite repeated transformation attempts. Therefore, the E10K, N15K, and V29W mutants were selected for subsequent experiments.

Bacteria harboring the correct sequences were cultured and induced for expression under the optimized conditions previously determined: 16°C, 0.5 mM IPTG, and 20 hours of induction. After induction, the cells were lysed, and the supernatant was purified. The purified product was incubated with lysis buffer for 4 hours. The reaction was terminated by pH adjustment, and the resulting material was collected for activity assays.

### Results:

The model generated multiple single-point mutant sequences, categorized into charge-enhanced and structure-optimized types. Charge-enhanced mutations primarily involved substitutions with positively charged residues, while structure-optimized mutations mainly featured small hydrophobic residues. The overall sequence framework remained conserved, with mutation sites predominantly located on the solvent-exposed face of the peptide chain, potentially favoring interfacial interactions.

Table 2. Binding Free Energy Results

| System | ΔVDW | ΔEEL | ΔG\_solv | ΔH | -TΔS | ΔG binding |
| --- | --- | --- | --- | --- | --- | --- |
| **WT + 4RHB** | -128.3 | -955.1 | 998.6 | -84.8 | 71.3 | -13.5 |
| **CecB-E10K + 4RHB** | -127.5 | -1102.3 | 1126.3 | -103.5 | 85.0 | -18.5 |
| **CecB-N15K + 4RHB** | -125.9 | -1180.7 | 1205.1 | -101.5 | 80.5 | -21.0 |
| **CecB-V29W + 4RHB** | -129.8 | -950.4 | 996.5 | -83.7 | 71.5 | -12.2 |
|  | | | | | | |
| **WT + 1DPE** | -95.6 | -680.2 | 715.3 | -60.5 | 50.1 | -10.4 |
| **CecB-E10K + 1DPE** | -93.8 | -695.1 | 729.4 | -59.5 | 50.0 | -9.5 |
| **CecB-N15K + 1DPE** | -90.1 | -705.8 | 742.5 | -53.4 | 45.0 | -8.4 |
| **CecB-V29W + 1DPE** | -145.2 | -675.5 | 720.8 | -99.9 | 82.3 | -17.6 |

The induced bacterial cultures appeared relatively clear, with minimal pellet observed after cell disruption. SDS-PAGE analysis of the expressed products revealed no distinct protein bands. Repeated expression attempts yielded no significant improvement. Furthermore, even after constructing double-point mutant plasmids, no corresponding protein bands or detectable antibacterial activity were observed.

### Learn:

Literature investigation into the P25A mutation revealed that proline at position 25 is a critical residue in Cecropin B, and its inherent structural rigidity likely makes substitutions at this site particularly challenging. For the other single-point mutants, none showed detectable expression. We hypothesize that the enhanced positive charge may compromise the protective efficacy of the anionic antioxidant peptide, leading to host toxicity and cell death. Subsequent attempts with double-point mutations also yielded unsatisfactory results.

Based on these findings, we concluded that while the native Cecropin B exhibits effective synergy with the anionic antioxidant peptide, our goal remains the design of a highly efficient antimicrobial peptide that confers minimal host toxicity.

## Discovering of New Peptide

### Introduction:

Based on preliminary experiments, Cecropin B didn't provide substantial benefits at this stage of our investigation. However, leveraging the rapid advancement of artificial intelligence, this study adopted a deep learning process to mine AMP (numbered 7, 4, 2) with brand-new sequence features from an extreme environment bacillus sequence library, and selected the optimal structural tandem configuration through molecular dynamics (MD) simulation. As a result, several monomers with potential performance, namely NJT-Lyy-7, NJT-Lyy-4, and NJT-Lyy-2, were obtained. For convenience, they will be referred to as "7", "4", and "2" in the following context.

### Design:

To build a robust antimicrobial peptide prediction model, we first integrated positive samples (AMPs) from multiple authoritative databases (such as APD3, DADP, DBAASP), and selected negative samples (non-AMPs) from the UniProtKB/Swiss-Prot database to construct a balanced dataset containing over 10,000 sequences. We adopted a bidirectional long short-term memory network (Bi-LSTM) with an attention mechanism as the core model architecture, and trained an integrated model through a five-fold cross-validation strategy to ensure its prediction accuracy and generalization ability.

After training, the integrated model was used to conduct a high-throughput scan on a genomic sequence library from extremophilic Bacillus. For sequences predicted by the model to have a high likelihood of being AMPs (prediction probability > 0.9), we applied a series of strict bioinformatics filtering conditions, including but not limited to assessing their potential to form amphipathic helices (hydrophobic moment), calculating net positive charge (range of +2 to +9), and excluding potential transmembrane sequences. Through this series of screening processes, three candidate AMP monomers with ideal characteristics were finally identified from the strain library and numbered as 7, 4, and 2, and used for subsequent experimental design and validation. Based on these results, we identified several candidate monomers with potential activity: NJT-Lyy-7, NJT-Lyy-4, and NJT-Lyy-2. For convenience, these are subsequently referred to as "7", "4", and "2".

The three monomers 7, 4, and 2 exhibited low solubility or unstable expression under various host/vector conditions. To enhance expression and folding stability, a strategy of tandemly linking the three monomers was adopted, with flexible (Gly4Ser)n linker peptides (length optimized through preliminary experiments) connecting the monomers in sequence to form a single polypeptide.

![Schematic diagram of the model structure](https://static.igem.wiki/teams/5809/engineering/engineering-32.webp)

Fig. 31 Schematic diagram of the model structure

Combinatorial space: All permutations of the three sites (A(3,3) = 6) were carried out to obtain six configurations: "742, 724, 472, 427, 274, 247"; for each configuration, the initial three-dimensional structure model and rapid energy minimization were conducted, and then the subsequent simulation screening was carried out (Fig. 32).

![RMSD curves from MD simulations](https://static.igem.wiki/teams/5809/engineering/engineering-33.webp)

Fig. 32 RMSD curves from MD simulations of the six tandem configurations

Then, using pET-28a(+) as the vector, a tandem expression cassette containing the pelB signal peptide sequence and codon-optimized antimicrobial peptides (AMP2, AMP4, and AMP7) was constructed, with the three peptides connected by a flexible linker (GGGGS). This recombinant plasmid was named pET-28a-pelB-742, or pET-28a-pelB-NJT-LYY-742.

![The structure of PelB-NJT-LYY-742](https://static.igem.wiki/teams/5809/engineering/engineering-34.webp)

Fig. 33 The structure of PelB-NJT-LYY-742

Plasmid of pET-28a-pelB-742 like Fig. 34

![Map of the pET-28a-pelB-742 plasmid](https://static.igem.wiki/teams/5809/engineering/engineering-35.webp)

Fig. 34 Map of the pET-28a-pelB-742 plasmid

![pET-28a-pelB-742 signaling pathway](https://static.igem.wiki/teams/5809/engineering/engineering-36.webp)

Fig. 35 pET-28a-pelB-742 signaling pathway

### Construction:

Similar to other DBTL constructions.

### Testing:

The correct colonies will be inoculated into two 50 mL LB media bottles. After reaching an OD600 of 0.6, they will be subjected to ultrasonic disruption. The protein from one of the bottles will be directly freeze-dried, while the other bottle will undergo purification and ultrafiltration before conducting an SDS-PAGE experiment. After the freeze-dried samples are melted, the inhibition zone test and MIC determination will be carried out.

The band size of pET-28a-pelB-NJT-LYY-742 is approximately 14.8 kDa.

![SDS-PAGE of PelB-742](https://static.igem.wiki/teams/5809/engineering/engineering-37.webp)

Fig. 36 SDS-PAGE image of PelB-742

**On the far right is the 10-180 kDa protein marker. Lane 1: Total protein of PelB-742; Lane 2: PelB-742 supernatant; Lane 3: PelB-742 precipitate; Lane 4: Sample after ultrafiltration of PelB-742**

![Antibacterial effect diagram of PelB-742](https://static.igem.wiki/teams/5809/engineering/engineering-38.webp)

Fig. 37 Antibacterial effect of PelB-742

![PelB-742 MIC Test Analysis Chart](https://static.igem.wiki/teams/5809/engineering/engineering-39.webp)

Fig. 38 PelB-742 MIC Test

### Result:

The experiment found that the antibacterial effect of PelB-742 has significantly improved, proving that our sequence combination is effective and functional. But the expression of 742 often in precipitate are not in supernatant.

### Learning:

Through experimental verification, it was found that the antibacterial effect of PelB combined with NJT-LYY-7, NJT-LYY-4, and NJT-LYY-2 is higher than that of individual proteins. However, when testing the antibacterial zone and MIC, we used the total protein for freeze-drying. However, the protein content we obtained in the supernatant was still relatively low. Therefore, we considered adding molecular chaperones to increase its expression in the supernatant.

## Molecular Chaperones

### Introduction:

Molecular chaperones are a class of proteins that can remove, degrade, and mark misfolded proteins to maintain protein homeostasis. They can promote the correct folding and soluble expression of proteins and effectively reduce the formation of inclusion bodies, without affecting the activity of the proteins. The Chaperone plasmid was selected to co-transform molecular chaperones and the pET-28a-NJT-LYY-742 plasmid into E. coli BL21 (DE3), enabling the chaperone protein group to work synergistically and participate in protein folding together, which can increase the recovery rate of soluble proteins.

### Design:

Four molecular chaperone plasmids, pGro7, pKJE7, pG-Tf2, and pTf16 (Table 3), were co-expressed with the pET-28a-NJT-LYY-742 plasmid to promote the correct folding of heterologous proteins and increase the solubility of heterologous proteins, in order to obtain a large amount of soluble proteins and thereby explore the biological function of this protein.

The modified strains were named pG-KJE8/742, pGro7/742, pKJE7/742, pG-Tf2/742, and pTf16/742.

Table 3. Molecular Chaperones

| Name | Companion protein | Promoter | Inducer | Resistance marker |
| --- | --- | --- | --- | --- |
| pGro7 | groES-groEL | araB | L-Arabinose | Cm |
| PKJE7 | dnaK-danJ-grpE | araB | L-Arabinose | Cm |
| pG-Tf2 | groES-groEL-tig | Pzt-1 | Tetracyclin | Cm |
| pTf16 | tig | araB | L-Arabinose | Cm |

### Construction:

Five molecular chaperone plasmids were separately transformed into the E. coli BL21 (DE3) host cells together with the pET-28a-pelB-NJT-LYY-742 plasmid through heat shock. The initial screening was conducted on plates containing Kan and Cm resistance. The single colonies with positive PCR results were induced and protein lysed for purification. After induction, the supernatant and precipitate obtained from cell disruption were subjected to SDS-PAGE.

### Test:

![SDS-PAGE co-expressed by chaperone and pET-28a-pelB-NJT-LYY-742](https://static.igem.wiki/teams/5809/result/resul-134.webp)

Fig. 39 SDS-PAGE co-expressed by chaperone and pET-28a-pelB-NJT-LYY-742

M: maker (10-180kDa); Lane 1: PG-Tf12/pelB-NJT-LYY-742-1 supernatant; Lane 2: PGro7/pelB-NJT-LYY-742-1 supernatant; Lane 3: PGro7/pelB-NJT-LYY-742-1 precipitate; Lane 4: pTf16/pelB-NJT-LYY-742-1 supernatant; Lane 5: pTf16/pelB-NJT-LYY-742-1 precipitate; Lane 6: PKJE7/pelB-NJT-LYY-742-1 supernatant; Lane 7: PKJE7/pelB-NJT-LYY-742-1 precipitate; Lane 8: pTf16/pelB-NJT-LYY-742-2 supernatant; Lane 9: pTf16/pelB-NJT-LYY-742-2 precipitate; Lane 10: PKJE7/pelB-NJT-LYY-742-2 supernatant; Lane 11: PKJE7/pelB-NJT-LYY-742-2 precipitate; Lane 12: PGro7/pelB-NJT-LYY-742-2 supernatant; Lane 13: PGro7/pelB-NJT-LYY-742-2 precipitate; Lane 14: PG-Tf12/pelB-NJT-LYY-742-2 supernatant

### Result:

We conducted SDS-PAGE experiments (Fig. 39) using the supernatant and precipitate. Through experimental verification, we found that molecular chaperones have a good effect on inclusion bodies. The bands in the supernatant were significantly higher than those without the addition of molecular chaperones. Moreover, we believe that the co-expression of PKJE7 and pelB-NJT-LYY-742 has the best effect.

### Learning:

Each molecular chaperone combination may act on a specific type of protein. In E. coli, it can effectively increase the number of chaperone proteins that enhance the soluble expression of heterologous proteins, and the induction conditions for expression vary depending on different target heterologous proteins. Therefore, in the subsequent studies, we will all make changes based on the strains containing molecular chaperones.

## Antibacterial Hydrogels

### Introduction:

Screen out formulations with antibacterial activity from three hydrogels with different basic formulations.

### Design:

Prepare and test the following three hydrogels:

1. Sodium Alginate/Gelatin Hydrogel (SOP 1)
2. PVA/Agarose Hydrogel (SOP 2)
3. Chitosan/Sodium Alginate Hydrogel (SOP 3, 1:1 ratio)

### Build:

The three hydrogels were prepared according to the following SOPs and made into circular gel sheets with a diameter of 6 mm for testing.

#### SOP 1: Sodium Alginate/Gelatin Double Network Hydrogel

1. Preparation of mixed solution: Dissolve 0.5 g of gelatin in 10 mL of water (60°C), then slowly add 0.2 g of sodium alginate and stir evenly.
2. Shaping and gelling: Pour the solution into a mold and place it in a 4°C refrigerator for 45 minutes to solidify the gelatin.
3. Crosslinking and washing: After demolding, immerse the gel in 2% CaCl₂‚ solution for 10 minutes, then rinse it with deionized water.

#### SOP 2: PVA/Agarose Physically CrossLinked Hydrogel

1. Preparation of mixed solution: Dissolve 1.0 g of PVA in 10 mL of water (95°C, 1-2 hours), then add 0.1 g of agarose and stir to dissolve.
2. Shaping and gelling: Pour the hot solution into a mold and cool it at room temperature for 30 minutes.
3. Freeze-­thaw cycle: Freeze the mold at -20°C for 12 hours and thaw it at room temperature for 2 hours. Repeat this cycle 3 times.

#### SOP 3: Chitosan/Sodium Alginate Polyelectrolyte Hydrogel Membrane

1. Preparation of solutions: Prepare 1% chitosan solution (dissolved in 1% acetic acid) and 1% sodium alginate aqueous solution respectively.
2. Electrostatic compounding: Under vigorous stirring, add the sodium alginate solution dropwise to the chitosan solution.
3. Film formation and hydration: Dry the mixture into a film in a petri dish, neutralize it with 1 M NaOH, rinse it thoroughly, and then allow it to swell into a gel in water.

### Test:

SOP 1 & SOP 2 hydrogels: No inhibition zone.

SOP 3 hydrogel (1:1): An obvious inhibition zone appeared (diameter≈12mm).

### Learn:

Only the formulation containing chitosan (SOP 3) has antibacterial activity. The next step will focus on optimizing this formulation.

## Optimization of Antibacterial Hydrogels

Optimize the component ratio of chitosan/sodium alginate hydrogel to enhance its antibacterial effect.

### Design:

Based on SOP 3, prepare three chitosan:sodium alginate hydrogels with different mass ratios:

1:2    1:1 (Control)    2:1

### Build:

Follow the SOP 3 process, only adjust the raw material mass to obtain samples with the above three ratios.

### Test:

Repeat the inhibition zone experiment.

![Inhibition zone](https://static.igem.wiki/teams/5809/engineering/engineering-41.webp)

Fig. 40 Inhibition zone

**P: 2:1; T: 1:1; N: 1:2**

1:2 ratio: Inhibition zone diameter≈8 mm.

1:1 ratio: Inhibition zone diameter≈12 mm.

2:1 ratio: The inhibition zone diameter is the largest, approximately 18 mm.

![The MIC of Hydrogels](https://static.igem.wiki/teams/5809/engineering/engineering-42.webp)

Fig. 41 The MIC of Hydrogels

### Learn:

Increasing the proportion of chitosan can significantly enhance the antibacterial effect. The ratio of chitosan to sodium alginate at 2:1 is the optimal formulation.

## Outlook

Currently, we have conducted preliminary investigations on Cecropin B and its modified variants. The engineered Cecropin B demonstrates certain antibacterial activity, with each variant showing improved efficacy to some extent. The expression of pET-28a-pelB-742 has been observed, indicating significant antibacterial activity. Furthermore, by incorporating molecular chaperones, we enhanced its soluble expression in the supernatant without markedly increasing the total yield, thereby improving the efficiency of utilizing this antimicrobial peptide. However, we believe that its performance can be further optimized. In subsequent stages, we plan to employ strategies such as solubility-enhancing tags and optimized promoters to achieve additional improvements.

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## Experiments

* [Preparation of LB Medium](#lb-medium)
* [Anion Antioxidant Peptide Protection Construction](#plasmid-cloning)
* [Gibson Assembly and Heat Shock Transformation](#gibson-assembly)
* [Product Purification](#product-purification)
* [Construction of Mutant Module Plasmid](#mutant-module)
* [Colony PCR and Sequencing](#colony-pcr)
* [Expand Cultivation, Induce](#expand-cultivation)
* [Bacterial Cell Collection Protein Extraction](#protein-extraction)
* [Protein Purification](#protein-purification)
* [SDS-PAGE Electrophoresis Verification](#sds-page)
* [EDPNG Cleavage Reaction System](#edpng-cleavage)
* [LYY Plasmid Module Construction](#lyy-construction)
* [Tandem Modules](#tandem-modules)
* [Bipartite Transformation](#bipartite-transformation)

## Preparation of LB Medium

Table 1 LB Culture Medium Formula

| Component | Concentration |
| --- | --- |
| Trypsin | 10 g/L |
| Sodium chloride | 10 g/L |
| Yeast | 5 g/L |

## Anion Antioxidant Peptide Protection Construction of Plasmid Cloning Fragments

Anion Antioxidant Peptide and PelB were cloned separately from plasmids stored in the laboratory. CecB was cloned from the synthesized pET-28a(+)-cecB plasmid.

Table 2 Primers Used in Experiments

| Primers | Sequence |
| --- | --- |
| CecB-R | CCTTTCGGGCTTTGTTAGCAG |
| CecB-F | GACCCGAACGGTatgAAGT |
| Yin-F | GAATTCGAGatgGATGCTCAAGAAAAACTAGAGATAGAAGC |
| ZT-F | CTGCTAACAAAGCCCGAAAGG |
| ZT-C-R | ACTTcatACCGTTCGGGTCGAGCTCGAATTCGGATCCG |
| ZT-Y-R | CTAGTTTTTCTTGAGCATCGAGCTCGAATTCGGATCCG |

Table 3 Reaction System

| Component | Volume |
| --- | --- |
| 2 × Phanta SE Buffer | 25 µL |
| Upstream primer (10 μM) | 2 µL |
| Downstream primer (10 μM) | 2 µL |
| Phanta SE Super-Fidelity DNA Polymerase | 1 µL |
| Template DNA | x µL |
| ddH2O | Up to 50 µL |

Table 4 Standard Reaction Procedure

| Loop steps | Temperature | Time | Number of loops |
| --- | --- | --- | --- |
| Pre-denaturation | 98℃ | 30 sec |  |
| Denaturation | 98℃ | 10 sec | 28-35 cycles |
| Annealing | 55℃ | 5 sec |  |
| Extend | 72℃ | 5 - 10 sec/kb |  |
| Completely extended | 72℃ | 1 min |  |

## Gibson Assembly and Heat Shock Transformation

1. Add appropriate fragments and carriers, add ddH2O to prepare the system to 5 µL, add 2 × CE Mix 5 µL. The process uses the Novagen ClonExpress Ultra One Step Cloning Kit V2 kit.
2. **Heat Shock Transformation:**
   * Take competent cells BL21 (DE3) out of the -80℃ freezer and place them on ice.
   * After competent cells thaw (about 5 min), add 10 µL plasmid to each 100 µL competent cells and incubate on ice for 30 min.
   * Heat shock at 42℃ for 90 seconds, then immediately take them out and place back on ice for 2-3 min.
   * In the heat-stimulated competent cells add 1 mL of antibiotic-free LB liquid medium and incubate on a 37℃ shaker for 1 hour.
   * Take 1 mL and mix the remaining 100 µL bacterial solution uniformly and spread it on LB solid medium containing the corresponding antibiotics.
   * Invert the LB solid medium and incubate in a constant temperature incubator at 37℃ overnight.

## Product Purification

Mix 10× DNA loading buffer with PCR amplification products at a 1:10 ratio, gently mix and perform 1% agarose gel electrophoresis using 1× TAE buffer. Run at approximately 30 min under 105 V. Observe the bands under a UV lamp to see if they match the size of the target fragment. Recover the target fragment based on gel operation. The DNA purification kit uses FastPureR Gel DNA Extraction Mini Kit (Nanjing Vazyme Enzyme Biotechnology Co., Ltd.), specific experimental steps are detailed in [Protocol]. Store the purified product in a -20℃ refrigerator for later use.

## Construction of Mutant Module Plasmid

Table 5 Primers Used in the Experiment

| Primer | Sequence |
| --- | --- |
| CecB-E10K-F | TAAAAAGATAaagAAAATGGGTCGTAATATTCGCAAC |
| CecB-E10K-R | GACCCATTTTcttTATCTTTTTAAATACTTTCCACTTcatACCG |
| CecB-N15K-F | ATGGGTCGTAAgATTCGCAACGGCAT |
| CecB-N15K-R | GTTGCGAATcTTACGACCCATTTTTTCTATCTTT |
| CecB-P25A-F | GAAGGCGGGTgctGCAATCGCCGTTTT |
| CecB-P25A-R | GGCGATTGCagcACCCGCCTTCACGATG |
| CecB-V29W-F | GCAATCGCCtggTTGGGCGAGGCGAAAG |
| CecB-V29W-R | TCGCCCAAccaGGCGATTGCCGGACC |

Table 6 Experimental Reaction System

| Component | Volume |
| --- | --- |
| 2 × Phanta SE Buffer | 25 µL |
| Upstream primer (10 μM) | 2 µL |
| Downstream primer (10 μM) | 2 µL |
| Phanta SE Super-Fidelity DNA Polymerase | 1 µL |
| Template DNA | x µL |
| ddH2O | Up to 50 µL |

Table 7 Specific Reaction Procedures

| Loop steps | Temperature | Time | Number of loops |
| --- | --- | --- | --- |
| Pre-denaturation | 98℃ | 30 sec |  |
| Denaturation | 98℃ | 10 sec | 28-35 cycles |
| Annealing | 55℃ | 5 sec |  |
| Extend | 72℃ | 5 - 10 sec/kb |  |
| Completely extended | 72℃ | 1 min |  |

PCR reaction is complete, add 1 μL Dpn I and 5 μL 10×EC Buffer for demethylation heat at 37°C for 40 min, then heat at 80°C for 20 min. After digestion, take 5 μL for AGE(agarose gel electrophoresis).

## Colony PCR

Signal peptide PelB and anionic antioxidant peptide colony PCR verification primers and systems are as follows, T7 and T7t are used to verify the presence of signal peptide part and the target gene of anionic antioxidant peptide on the plasmid.

Table 8 Primers Used in the Experiment

| Primers | Sequence |
| --- | --- |
| T7 | TAATACGACTCACTATAGGG |
| T7t | GCTAGTTATTGCTCAGCGG |

Table 9 Colony PCR Reaction System

| Composition | Volume |
| --- | --- |
| 2×Rapid Taq Master Mix | 5 µL |
| Upstream Primer (10 µM) | 0.4 µL |
| Downstream Primer (10 µM) | 0.4 µL |
| Colony | Not counted |
| ddH2O | Up to 10 µL |

1. Prepare the PCR reaction mixture according to the above system.
2. From multiple single colonies selected from the culture dishes after transformation, immerse them separately into the reaction systems of sterile eight-tube racks.
3. After the reaction is complete and verified through AGE(agarose gel electrophoresis). If fragments of the corresponding size are present, picking the single colony for culturing, then transfer it to liquid medium. Store the strains in a 1:1 glycerol and bacterial solution, and the rest can be sent for sequencing.

## Expand Cultivation, Induce

1. After successful sequencing, place the reserved strains in a liquid culture medium containing 5 mL LB (50 µg/mL Kan) in a shaking tube, and incubate for about 5 hours.
2. Take 1-2 mL of bacterial liquid from the shaking tube, and transfer it to a flask containing 50 mL LB (50 µg/mL Kan).
3. Shake at 37℃, 200 rpm until OD600=0.6
4. Add IPTG with a final concentration of 0.5 mM to the flask on a clean bench.
5. 18℃, Incubate at 200 rpm for 20 h.

## Bacterial Cell Collection Protein Extraction

1. **Cell collection:** After induction, centrifuging the culture at 8000 rpm for 5 min, discard the supernatant. Add 10 mL of PBS with pH 7.0 to the centrifuge tube, suspend the cells to wash away the medium, and centrifuge again at 8000 rpm for 5 min, discard the supernatant. Repeat this step 3 times, then add 10 mL of PBS with pH 7.0 to resuspend the cells.
2. Place the centrifuge tube in an ultrasonicator with a power of approximately 250 W, ultrasonicate for 3 s followed by a 5 s interval, for a total of 10 min. Utilize the high-frequency vibration of the ultrasound to break down the cell walls until the bacterial liquid becomes clear.
3. After ultrasonication, centrifuge at 4°C, 12000 rpm for 30 min to separate the supernatant and precipitate.

## Protein Purification

1. Prepare the nickel column (stored in 20% alcohol), wait for the alcohol to evaporate, and sequentially add 5 mL Buffer A (20 mM imidazole), 3 mL Buffer B (250 mM imidazole), 5 mL Buffer A to wash the column.
2. Add the crude enzyme solution and repeat the adsorption 3 times, wash with 5 mL Buffer A to remove impurity proteins. Then add 5 mL Buffer B and pour into the pre-marked beaker to obtain the target protein.
3. Separately add 5 mL Buffer B, 5 mL Buffer A, 5 mL H2O clean the nickel column, and seal the column with 20% alcohol.
4. Pour the crude enzyme solution from the beaker into the ultrafiltration tube (sealed with 20% alcohol), and centrifuge at 4℃, 6500 rpm for 30 min. If the final concentrated volume of the pure enzyme solution exceeds 500~700 µL, centrifuge for an additional 5~15 min to increase protein concentration.
5. After completion, aspirate the upper layer and rinse the ultrafiltration tube with up water.
6. Fill the ultrafiltration tube with 0.1 mM NaOH, and soak at 4℃ for 1 h. Then centrifuge at 6500 rpm for 15 min.
7. The pure enzyme solution after concentration is stored in a -20℃ freezer, and the ultrafiltration tubes are preserved with 20% alcohol.

## SDS-PAGE Electrophoresis Verification

According to the SDS-PAGE electrophoresis method in the [protocol], samples are prepared based on protein concentration, and SDS-PAGE electrophoresis is performed on the supernatant, precipitate, and purified enzyme solution to observe whether bands corresponding to the target protein are present.

## EDPNG Cleavage Reaction System

Under the condition of a volume ratio of 2:1, the lysis solution was added to all purified liquids. The lysis solution included: 1.86 M hydroxylamine hydrochloride solution and 100 mM Tris-HCl, pH=9.0. After the reaction system was prepared, it was placed at 45℃ for 4 hours of reaction. After the reaction was completed, 1 M HCl solution was added to adjust the pH to 7.0 to terminate the reaction. After the reaction was terminated for a period of time, all reaction solutions were added to the ultrafiltration tube to obtain the upper liquid for subsequent experiments.

## LYY Plasmid Module Construction

### Signal Peptide Section

Signal peptide PelB comes from pET-28a(+)-pelB-yin-cecB. LYY-7, LYY-4, LYY-2 come from plasmids synthesized by the company (General Biol).

Table 10 Primers Used in the Experiment

| Primers | Sequence |
| --- | --- |
| ZT-R | CGCCATTGCCGGCT |
| NJT-LYY-7-F | CTGCGCAGCCGGCAATGGCGTGGACACCCGCTCTATC |
| NJT-LYY-7-R | GCTTCTTTCGCAGCTGCCTCGCACGCACGGGTG |
| ZT-F | GAGGCAGCTGCGAAAG |
| NJT-LYY-4-F | CTGCGCAGCCGGCAATGGCGAACGATGTAAATCCAGAAACTACAC |
| NJT-LYY-4-R | GCTTCTTTCGCAGCTGCCTCACAACGAGAGGTGCACTTG |
| NJT-LYY-2-F | CTGCGCAGCCGGCAATGGCGTGTACTACAAATACGTTTAGTCTATCAGATTACTG |
| NJT-LYY-2-R | GCTTCTTTCGCAGCTGCCTCGCACCACGCCATGCAC |

Table 11 Experimental Reaction System

| Component | Volume |
| --- | --- |
| 2 × Phanta SE Buffer | 25 µL |
| Upstream primer (10 μM) | 2 µL |
| Downstream primer (10 μM) | 2 µL |
| Phanta SE Super-Fidelity DNA Polymerase | 1 µL |
| Template DNA | x µL |
| ddH2O | Up to 50 µL |

Table 12 Specific Reaction Procedures

| Loop steps | Temperature | Time | Number of loops |
| --- | --- | --- | --- |
| Pre-denaturation | 98℃ | 30 sec |  |
| Denaturation | 98℃ | 10 sec | 28-35 cycles |
| Annealing | 55℃ | 5 sec |  |
| Extend | 72℃ | 5 - 10 sec/kb |  |
| Completely extended | 72℃ | 1 min |  |

PCR reaction is complete, add appropriate fragments and vector, add ddH2O to prepare the system to 5 µL, add 2 × CE Mix 5 µL. The process uses the Novozyme ClonExpress Ultra One Step Cloning Kit V2 kit for recombination transformation.

### Colony PCR and Sequencing

PelB-NJT-LYY primers and systems for verification are as follows, T7 and T7t are used to verify the presence of signal peptide part and the target gene of anionic antioxidant peptide on the plasmid.

Table 13 Primer Used in the Experiment

| Primers | Sequence |
| --- | --- |
| T7 | TAATACGACTCACTATAGGG |
| T7t | GCTAGTTATTGCTCAGCGG |

Table 14 Culture PCR Reaction System

| Composition | Volume |
| --- | --- |
| 2×Rapid Taq Master Mix | 5 µL |
| Upstream Primer (10 µM) | 0.4 µL |
| Downstream Primer (10 µM) | 0.4 µL |
| Colony | Not counted |
| ddH2O | Up to 10 µL |

1. Prepare the PCR reaction mixture according to the above system.
2. From the culture plates incubated overnight after transformation, select multiple single colonies and immerse them separately in reaction systems with sterile tips.
3. After the reaction is complete and verified via AGE(agarose gel electrophoresis), if fragments of the corresponding size are observed, streak the colony plates for further culturing, then transfer them to liquid medium. Preserve the strains with 50% glycerol at a 1:1 ratio, and the remaining ones can be sent for sequencing.

## Tandem Modules

The predicted combinations were constructed to obtain PelB-NJT-LYY-742.

Table 15 Primers Used in the Experiment

| Primer | Sequence |
| --- | --- |
| NJT-LYY-7-R | GCTTCTTTCGCAGCTGCCTCGCACGCACGGGTG |
| ZT-F | GAGGCAGCTGCGAAAG |
| 7&4-F | GCACCCGTGCGTGCGAGCTCAACGATGTAAATCCAGAAACTACAC |
| NJT-LYY-4-R | GCTTCTTTCGCAGCTGCCTCACAACGAGAGGTGCACTTG |
| 4&2-F | GCACCTCTCGTTGTGTCGACTGTACTACAAATACGTTTAGTCTATCAGATTACTG |
| NJT-LYY-2-R | GCTTCTTTCGCAGCTGCCTCGCACCACGCCATGCAC |

Table 16 Experimental Reaction System

| Component | Volume |
| --- | --- |
| 2 × Phanta SE Buffer | 25 µL |
| Upstream primer (10 μM) | 2 µL |
| Downstream primer (10 μM) | 2 µL |
| Phanta SE Super-Fidelity DNA Polymerase | 1 µL |
| Template DNA | x µL |
| ddH2O | Up to 50 µL |

Table 17 Specific Reaction Procedure

| Loop steps | Temperature | Time | Number of loops |
| --- | --- | --- | --- |
| Pre-denaturation | 98℃ | 30 sec |  |
| Denaturation | 98℃ | 10 sec | 28-35 cycles |
| Annealing | 55℃ | 5 sec |  |
| Extend | 72℃ | 15 sec/kb |  |
| Completely extended | 72℃ | 1 min |  |

PCR reaction is complete after, add appropriate fragments and vectors, add ddH2O to prepare the system to 5 µL, add 2 × CE Mix 5 µL. Process uses Novagen ClonExpress Ultra One Step Cloning Kit V2 kit for recombination heat shock transformation and sends to the company for sequencing.

## Bipartite Transformation

1. Mix the molecular chaperone plasmid with pET-28a(+)-cecB plasmid in equal amounts.
2. Heat shock transformation:
   * Take the competent cells BL21 (DE3) out of the -80℃ freezer and place them on ice.
   * Competent cells after thawing (about 10 min), add 10 µL of the mixed plasmid to each 100 µL of competent cells and chill on ice for 30 min.
   * Heat shock at 42℃ for 90 seconds, immediately transfer back to ice and incubate for 2-3 min.
   * Add 700 µL of antibiotic-free LB liquid medium to the heat-shocked competent cells and incubate on a shaker at 37℃ for 1 hour.
   * Take 100 µL of the bacterial solution and spread it on LB solid medium containing the corresponding antibiotic.
   * Invert the solid medium and incubate overnight at 37℃ in a constant temperature incubator.
3. After heat shock transformation, use LB plates containing dual resistance (Cm, Kan) for initial screening. Invert the plates and incubate at 37℃ for 12 h to verify colony selection.

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## Results

* [Simulation and prediction](#simulation)
* [Improved antibacterial performance](#improved)
* [Mutated section](#mutated)
* [Discovering of Novel peptide](#novel)
* [Molecular chaperones](#chaperones)
* [Hydrogel](#hydrogel)

## Simulation and prediction

We performed sequence analysis by conducting BLASTp searches in NCBI's non-redundant protein sequence database. After removing non-clustered sequences, we optimized the codons. Subsequently, Alphafold2 was employed to model the structural conformations of these sequences.

![The structure prediction diagram of cecropin B](https://static.igem.wiki/teams/5809/result/result111.webp)

Fig.1 The structure prediction diagram of cecropin B

## Improved antibacterial performance

Using the IGEM Registry, we selected CecB as our antimicrobial peptide sequence. Through NCBI's BLASTp database, we compared the protein sequences obtained from the IGEM Registry. Subsequently, we optimized its codon usage using the company's codon optimization software. The synthesized primer was ligated into a CecB fragment and inserted into the pET-28a vector for expression, which served as the bacterial host for subsequent cultivation.

### Plasmid design:

![pET-28a-cecB](https://static.igem.wiki/teams/5809/result/result112.webp)

Fig. 2 pET-28a-cecB

![pET-28a-yin-cecB](https://static.igem.wiki/teams/5809/result/result113.webp)

Fig. 3 pET-28a-yin-cecB

![pET-28a-pelB-yin-cecB](https://static.igem.wiki/teams/5809/result/result114.webp)

Fig. 4 pET-28a-pelB-yin-cecB

All the primers were put into the PCR system for amplification to synthesize fragments without template, and the correct fragments were recovered from the gel.

We extracted the plasmid from the synthesized gene and transformed it into E.coli BL21 (DE3). The transformed single colony was selected and cultured on the plate, and the bacterial colony PCR was used to verify whether it contained the recombinant plasmid.

![cecB colony PCR image](https://static.igem.wiki/teams/5809/result/result115.webp)

Fig. 5 cecB colony PCR

![yin-cecB colony PCR image](https://static.igem.wiki/teams/5809/result/result116.webp)

Fig. 6 yin-cecB colony PCR

![pelB-yin-cecB bacterial colony PCR image](https://static.igem.wiki/teams/5809/result/result117.webp)

Fig. 7 pelB-yin-cecBcolony PCR

After culturing the colonies with confirmed results, the activated bacterial suspension was added to 50 mL LB medium for induction. Following the induction process, the cells were homogenized and the supernatant was purified through ultrafiltration. Total protein content, supernatant liquid, and sediment were analyzed using Western blot (WB) experiments (Fig.8,9) to determine their inhibition zones and Minimum Inhibitory Concentration (MIC)(Fig.10,11,12).

After induction, the supernatant and precipitate of cell pulverization were respectively subjected to SDS-PAGE

![cecB WB image](https://static.igem.wiki/teams/5809/result/result118.webp)

Fig. 8 CecB WB image

![pelB-yin-cecB WB image](https://static.igem.wiki/teams/5809/result/result119.webp)

Fig. 9 PelB-Yin-CecB WB image

The leftmost lane is 10-180kDa Marker, lane 1 is ultrafiltration protein sample, lane 2 is total protein sample, and lane 3 is precipitation protein (using 5mL PBS pH=7.4 to resuspend the precipitation)

We found that the effect of direct lyophilization was better than that of purified ultrafiltration. Therefore, the crushed protein was added to the lysis solution with twice its volume to break the anion antioxidant peptide, and CecB protein was obtained after freeze-drying for MIC and inhibition effect determination.

![Results of the inhibition circle experiment](https://static.igem.wiki/teams/5809/result/result120.webp)

Fig. 10 Results of the inhibition circle experiment

1：PelB-Yin-CecB；2: Yin-CecB-1；3：CecB；4：Yin-CecB-2

After determination, it was found that the target band could not be obtained in WB, and no obvious inhibitory effect of CecB was found in both inhibition zone and MIC results.

![yin-cecB MIC experiment](https://static.igem.wiki/teams/5809/result/result121.webp)

Fig. 11 Yin-CecB MIC experiment

![pelB-yin-cecB MIC](https://static.igem.wiki/teams/5809/result/result122.webp)

Fig. 12 PelB-Pin-CecB MIC

After determination, it was found that the target band could not be obtained in WB, and no obvious inhibitory effect of CecB was found in both inhibition zone and MIC results.

### Fermentation optimization

We investigated the fermentation temperature of the above PelB-Yin-CecB protein. The fermentation was carried out in LB medium, and 16℃ 25℃ 30℃ 37℃ of the fermentation broth was taken respectively for crushing and purification, protein concentration was determined, SDS-PAGE was used to verify the expression and optical density was determined.

Since the concentration of IPTG is also one of the important factors affecting the expression during induction, we induced the protein at a gradient of 0.25 mM, 0.5 mM and 1 mM. The fermentation broth was broken and purified to obtain the protein-like liquid for testing the optical density of the SDS-PAGE.

To explore the effect of culture medium on CecB, we also used TB culture medium and LB culture medium as control.

![Optical density image of temperature](https://static.igem.wiki/teams/5809/result/result123.webp)

Fig. 13 Optical density image of temperature

![Optical density image of temperature](https://static.igem.wiki/teams/5809/result/result124.webp)

Fig. 14 Optical density image of temperature

![Optical Density image of IPTG Concentration](https://static.igem.wiki/teams/5809/result/result125.webp)

Fig. 15 Optical Density image of IPTG Concentration

![Optical Density image of IPTG Concentration](https://static.igem.wiki/teams/5809/result/result126.webp)

Fig. 16 Optical Density image of IPTG Concentration

![Optical density image of media optimization](https://static.igem.wiki/teams/5809/result/result127.webp)

Fig. 17 Optical density image of media optimization

![Optical density image of media optimization](https://static.igem.wiki/teams/5809/result/result128.webp)

Fig. 18 Optical density image of media optimization

According to the optical density image, when the temperature is about 16℃, more bands can be obtained in the supernatant. The percentage image shows that the protein content in the supernatant is higher, so we choose 16℃ for induction of expression in the following.

According to the optical image of IPTG concentration test, when the OD600 of bacterial solution reached 0.6, IPTG was added for induction to make the final concentration of 0.5 mM.

Through optimized medium cultivation, we observed that when pelB was added, the natural content of silkworm toxin in the precipitate became minimal. However, with TB medium, the toxin was fully detected in the supernatant. Notably, in plasmid without signal peptides, protein levels in the supernatant of TB medium also increased. Yet, when freeze-drying without ultrafiltration, no significant antibacterial effects were observed a situation that not only wastes time and financial resources but also contradicts our research expectations.

## Mutated section

The pre-trained large language model is utilized for task-oriented fine-tuning. The fine-tuning dataset consists of known antimicrobial peptide sequences and their functional annotations, aiming to enhance the model's recognition capabilities for key physicochemical characteristics (charge distribution, amphiphilicity, secondary structure propensity). Charge-enhancing modifications: By replacing positively charged residues such as Lys/Arg, interactions with negatively charged targets are enhanced;

Structural optimization: improve the folding and groove compatibility by replacing hydrophobic or small volume residues.

All candidate sequences are standardized and organized into a table file for subsequent calculation and verification.

The model generated multiple single-point mutation sequences, categorized as charge-enhancing and structure-optimizing types. Charge-enhancing mutations predominantly involved positive residue substitutions, while structure-optimizing mutations mainly featured small-volume hydrophobic residue replacements. The overall sequence retained its original backbone structure, with mutation sites distributed along the peptide chain's outer side to facilitate interfacial interactions. The binding free energy of these mutants to bacterial receptor proteins was also calculated.

Table 1. Results combined with free energy

| System | ΔVDW | ΔEEL | ΔG\_solv | ΔH | -TΔS | End G binding |
| --- | --- | --- | --- | --- | --- | --- |
| WT + 4RHB | -128.3 | -955.1 | 998.6 | -84.8 | 71.3 | -13.5 |
| CecB-E10K + 4RHB | -127.5 | -1102.3 | 1126.3 | -103.5 | 85.0 | -18.5 |
| CecB-N15K + 4RHB | -125.9 | -1180.7 | 1205.1 | -101.5 | 80.5 | -21.0 |
| CecB-V29W + 4RHB | -129.8 | -950.4 | 996.5 | -83.7 | 71.5 | -12.2 |
|  | | | | | | |
| WT + 1DPE | -95.6 | -680.2 | 715.3 | -60.5 | 50.1 | -10.4 |
| CecB-E10K + 1DPE | -93.8 | -695.1 | 729.4 | -59.5 | 50.0 | -9.5 |
| CecB-N15K + 1DPE | -90.1 | -705.8 | 742.5 | -53.4 | 45.0 | -8.4 |
| CecB-V29W + 1DPE | -145.2 | -675.5 | 720.8 | -99.9 | 82.3 | -17.6 |

After induction, the bacterial cells were relatively clear, and there was almost no precipitation after fragmentation. Moreover, it was verified by SDS-PAGE that there was no obvious band. Subsequent corresponding expression results showed no obvious effect. Although we still constructed the double-point mutation plasmid in the later stage, no relevant band or effect was found.

## Discovering of Novel peptide

Based on preliminary experiments, Cecropin B didn't provide substantial benefits at this stage of our investigation. However, leveraging the rapid advancement of artificial intelligence, this study adopted a deep learning process to mine AMP (numbered 7, 4, 2) with brand-new sequence features from an extreme environment bacillus sequence library, and selected the optimal structural tandem configuration through molecular dynamics (MD) simulation. As a result, several monomers with potential performance, namely NJT-Lyy-7, NJT-Lyy-4, and NJT-Lyy-2, were obtained. For convenience, they will be referred to as "7", "4", and "2" in the following context.

We first integrated positive samples (AMPs) from multiple authoritative databases (such as APD3, DADP, DBAASP), and selected negative samples (non-AMPs) from the UniProtKB/Swiss-Prot database to construct a balanced dataset containing over 10,000 sequences. We adopted a bidirectional long short-term memory network (Bi-LSTM) with an attention mechanism as the core model architecture, and trained an integrated model through a five-fold cross-validation strategy to ensure its prediction accuracy and generalization ability.

After training, the integrated model was used to conduct a high-throughput scan on a genomic sequence library from extremophilic Bacillus.

All permutations of the three sites (A(3,3) = 6) were carried out to obtain six configurations: "742, 724, 472, 427, 274, 247"; for each configuration, the initial three-dimensional structure model and rapid energy minimization were conducted, and then the subsequent simulation screening was carried out. (Fig.19)

![RMSD curves from MD simulations of the six tandem configurations](https://static.igem.wiki/teams/5809/result/result129.webp)

Fig.19 RMSD curves from MD simulations of the six tandem configurations

Then, using pET-28a(+) as the vector, a tandem expression cassette containing the pelB signal peptide sequence and codon-optimized antimicrobial peptides (AMP2, AMP4, and AMP7) was constructed, with the three peptides connected by a flexible linker (GGGGS). This recombinant plasmid was named pET-28a-pelB-742, or pET-28a-pelB-NJT-LYY-742.

![Map of the pET-28a-pelB-742 plasmid](https://static.igem.wiki/teams/5809/result/result130.webp)

Fig. 20 Map of the pET-28a-pelB-742 plasmid

The correct colonies will be inoculated into two 50 mL LB media bottles. After reaching an OD600 of 0.6, they will be subjected to ultrasonic disruption. The protein from one of the bottles will be directly freeze-dried, while the other bottle will undergo purification and ultrafiltration before conducting an SDS-PAGE experiment. After the freeze-dried samples are melted, the inhibition zone test and MIC determination will be carried out.

The band size of pET-28a-pelB-NJT-LYY-742 is approximately 14.8 kDa. (Fig.21)

![pelB-742 SDS-PAGE](https://static.igem.wiki/teams/5809/result/result131.webp)

Fig.21 pelB-742 SDS-PAGE image

n the far right is the 10-180 kDa protein marker. Lane 1: Total protein of PelB-742; Lane 2: PelB-742 supernatant; Lane 3: PelB-742 precipitate; Lane 4: Sample after ultrafiltration of PelB-742

![pelB-742 Antimicrobial effect](https://static.igem.wiki/teams/5809/result/result132.webp)

Fig. 22 pelB-742 Antimicrobial zone

![pelB-742MIC Test analysis diagram](https://static.igem.wiki/teams/5809/result/result133.webp)

Fig. 23 pelB-742 MIC Test

The experimental results showed that the antibacterial effect of pET-28a-pelB-742 was significantly improved, which proved that our series was effective and effective. The results also showed that when the concentration of antibacterial peptide was close to 3.13 µg/mL, it still had antibacterial effect on the indicator bacteria.

## Molecular chaperones

Molecular partners are a class of proteins that can remove, degrade and label misfolded proteins to maintain protein homeostasis. They can promote the correct folding and soluble expression of proteins, effectively reduce the formation of inclusions, but do not affect the activity of proteins.

Four kinds of molecular chaperone plasmids, pGro7, pKJE7, pG-Tf2 and pTf16 were co-expressed with pET-28a-NJT-LYY-742 plasmid to promote the correct folding of foreign protein in prokaryotic cells and increase the solubility of foreign protein, so as to obtain a large amount of soluble protein and further explore the biological function of the protein.

Four molecular chaperone plasmids were co-transfected into Escherichia coli BL21(DE3) host cells with the pET-28a-pelB-NJT-LYY-742 plasmid. Primary screening was conducted on Kan and Cm antibiotic-resistant plates. Single colonies PCR-positive were selected for induction and protein extraction purification. After induction, cell homogenized supernatants and precipitates underwent SDS-PAGE (Fig.24) for analysis.

![SDS-PAGE co-expressed by chaperone and pET-28a-pelB-NJT-LYY-742](https://static.igem.wiki/teams/5809/result/resul-134.webp)

Fig. 24 SDS-PAGE co-expressed by chaperone and pET-28a-pelB-NJT-LYY-742

M:maker(10-180kDa); Lane 1:PG-Tf12/pelB-NJT-LYY-742-1 supernatant; Lane 2: PGro7/pelB-NJT-LYY-742-1 supernatant; Lane 3:PGro7/pelB-NJT-LYY-742-1 precipitate; Lane 4: pTf16/pelB-NJT-LYY-742-1 supernatant; Lane 5:pTf16/pelB-NJT-LYY-742-1 precipitate; Lane 6:PKJE7/pelB-NJT-LYY-742-1 supernatant; Lane 7:PKJE7/pelB-NJT-LYY-742-1 precipitate; Lane 8:pTf16/pelB-NJT-LYY-742-2 supernatant; Lane 9:pTf16/pelB-NJT-LYY-742-2 precipitate; Lane 10:PKJE7/pelB-NJT-LYY-742-2 supernatant; Lane 11:PKJE7/pelB-NJT-LYY-742-2 precipitate; Lane 12:PGro7/pelB-NJT-LYY-742-2 supernatant; Lane 13:PGro7/pelB-NJT-LYY-742-2 precipitate; Lane 14:PG-Tf12/pelB-NJT-LYY-742-2 supernatant

We conducted SDS-PAGE experiments (Fig.24) using the supernatant and precipitate. Through experimental verification, we found that molecular chaperones have a good effect on inclusion bodies. The bands in the supernatant were significantly higher than those without the addition of molecular chaperones. Moreover, we believe that the co-expression of PKJE7 and pelB-NJT-LYY-742 has the best effect.

## Hydrogel

Antimicrobial formulations were selected from three different basic hydrogel formulations. The following three hydrogels were prepared and tested:

Sodium alginate/gelatin hydrogel (SOP 1)

PVA/agarose hydrogel (SOP 2)

The hydrogel of chitosan/sodium alginate (SOP 3, 1:1 ratio) was made into circular gel sheet with a diameter of 6 mm for testing. It was found that only SOP 3 had effective effect. Therefore, the composition ratio of chitosan/sodium alginate hydrogel was optimized to enhance its antibacterial effect.

Based on SOP 3, three different mass ratios of chitosan were prepared: sodium alginate hydrogel: 1:2, 1:1(Control), 2:1

In accordance with SOP 3 process, only the raw material quality was adjusted to obtain the above three proportions of samples.

Repeated inhibition circle experiment.

![Inhibitory circle test](https://static.igem.wiki/teams/5809/result/result-135.webp)

Fig 25. Inhibitory circle test

P：2:1 T: 1:1 N: 1:2

1:2 ratio: the diameter of the antibacterial circle is about 8 mm.

1:1 ratio: the diameter of the antibacterial circle is about 12 mm.

2:1 ratio: the maximum diameter of the antibacterial circle is about 18 mm.

![The MIC of Hydrogels](https://static.igem.wiki/teams/5809/result/result-136.webp)

Fig. 26. The MIC of Hydrogels

Increasing the proportion of chitosan can significantly enhance the antibacterial effect. The effect of 2:1 can reach 6.25 mg/mL. The optimal formula is 2:1 ratio of chitosan and sodium alginate.

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