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).

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).

Fig. 2 The structure prediction diagram of 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.

Fig. 3 Results of NCBI sequence alignment

The plasmid map and signal pathway are illustrated as follows:

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

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.

Fig. 6 Colony PCR results of cecB

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.

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.

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.

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).

Fig. 10 pET-28a-yin-CecB

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).

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.

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).

Fig. 14 Inhibition assay results of Yin-CecB

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

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.

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.

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

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.

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:

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).

Fig. 20 PelB-Yin-CecB MIC

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.

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.

Fig. 22 Optical density image of temperature

Fig. 23 Optical density image of temperature

Fig. 24 Optical density image of IPTG Concentration

Fig. 25 Optical density image of IPTG Concentration

Fig. 26 Optical density image of media optimization

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.

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.

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.

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.

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.

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.

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).

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.

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

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

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

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.

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

Fig. 37 Antibacterial effect of PelB-742

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.

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:

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.

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.

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.

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.

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.

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.