June 12-20, 2025

An antimicrobial peptide (AMP) development project was initiated. Through searches in the iGEM Part Registry and literature reviews, focus was directed to Cecropin B (CecB), which exhibits broad-spectrum antibacterial activity and low toxicity toward eukaryotic cells. Due to the high cost of chemical synthesis of AMPs, a recombinant biosynthesis approach was adopted. However, the inherent toxicity of CecB to the E. coli expression host posed challenges during expression. Sequence alignment and molecular docking predictions were subsequently performed.

June 20-27, 2025

The CecB amino acid sequence was aligned using NCBI BLASTp, and the NCBI non-redundant protein database was searched to confirm sequence conservation. The gene sequence was codon-optimized for the E. coli expression system. The optimized CecB gene was imported into SnapGene to construct the recombinant plasmid pET-28a-cecB. Based on this template, primers were designed and synthesized commercially for a synthesis experiment.

June 27-July 3, 2025

Synthesis was performed using the synthesized primers to obtain the CecB gene sequence, which was then cloned into the pET-28a plasmid to construct the recombinant plasmid pET-28a-cecB. This plasmid was transformed into E. coli DH5α, plated on kanamycin-containing LB plates, and incubated at 37°C for 16 hours. Colony PCR was conducted to verify the correct colonies. A confirmed single colony was expanded in 5 mL of culture medium and sent to a company for sequencing confirmation. The sequence-verified plasmid was then heat-shock transformed into the expression host E. coli BL21(DE3) to obtain the recombinant engineered strain.

July 5-8, 2025

After selection and activation of single colonies, cultures were inoculated into 50 mL of LB medium (containing kanamycin at 50 μg/mL) and grown at 37°C until OD₆₀₀ reached ~0.6. IPTG was added to a final concentration of 0.5 mM, and induction was carried out at 18°C for 16 hours. Cells were collected, sonicated, and supernatant and pellet were separated. Western blot analysis of the expressed product showed no detectable CecB band at the 5.4 kDa position.

July 8-9, 2025

Using E. coli and Bacillus subtilis as indicator strains, antibacterial activity was tested using the agar well diffusion method. Antibacterial assays were conducted on the crude protein extract, and its minimum inhibitory concentration (MIC) was determined.

July 10-13, 2025

To reduce CecB toxicity to the host and promote soluble expression, a fusion expression strategy was designed: an anionic antioxidant peptide was fused to the N-terminus of CecB to neutralize its positive charge. Using SnapGene software, the fusion tag was designed to connect with the pET-28a-cecB vector, generating the recombinant plasmid pET-28a-yin-cecB. Corresponding primers were designed and sent for synthesis to confirm the template plasmid.

July 14-21, 2025

The fusion tag fragment was amplified via PCR and ligated with the linearized pET-28a-cecB vector to construct the recombinant plasmid pET-28a-yin-cecB, which was transformed into E. coli DH5α. After transformation, colony picking, PCR verification, and sequencing were performed. The correct plasmid was transformed into E. coli BL21(DE3). Selected colonies were verified, and the bacteria were activated.

July 21-25, 2025

Activated bacteria were inoculated into 50 mL of LB medium (with kanamycin at 50 μg/mL), grown at 37°C until OD₆₀₀~0.6, induced with 0.5 mM IPTG, and expressed at 18°C for 20 hours. Bacterial cells were collected, lysed, and centrifuged to obtain the supernatant. Twice the volume of lysis buffer was added to the purified protein and incubated at 45°C for 4 hours. The protein was then obtained by ultrafiltration. Cecropin B expression was analyzed by SDS-PAGE, and antibacterial activity was tested.

July 25-30, 2025

Repeat the above steps to select points again for activation-induced expression and test their antibacterial activity.

July 31-August 2, 2025

For construction of plasmids with signal peptide PelB, the signal peptide linked to pET-28a-yin-cecB was designed using SnapGene. Corresponding primers were designed, and synthesis was commissioned to confirm the template plasmid pET-28a-yin-cecB.

August 10-11, 2025

The signal peptide fragment and the linearized pET-28a-yin-cecB vector were obtained through PCR amplification, and the amplified fragments were verified by nucleic acid gel electrophoresis. After confirmation, fragments were recovered from the gel. The concentrations of the recovered fragments and vector were measured and stored at -20°C for subsequent use. Using one-step cloning, the fragments and vector were recombined to construct pET-28a-pelB-yin-cecB. The recombinant plasmid was transformed via heat shock and cultured. A portion of the bacterial culture was spread onto LB plates containing antibiotics for preliminary screening. Plates were inverted and incubated at 37°C for 12 hours.

August 12-14, 2025

Five to seven single colonies were selected from each plate and verified via colony PCR. Correctly verified colonies were transferred into 5 mL tubes containing antibiotics and cultured for 12 hours. Then, 1 mL of bacterial culture was sent for sequencing.

August 15-22, 2025

The correctly sequenced strains were preserved and cultured on a larger scale. At OD₆₀₀ = 0.6, 250 μL of 0.1 mM IPTG was added, and induction was performed at 18°C for 20 hours. After induction, bacteria were collected, ultrasonically disrupted, and centrifuged to collect the supernatant. After purification and a 4-hour lysis reaction, the protein was obtained by ultrafiltration. Protein expression was determined by SDS-PAGE and the band density was measured. Re-induced the recombinant plasmids with incorrect protein bands and extracted the proteins.

July 20, 2025-July 25, 2025

Using E. coli BL21(DE3)/pET-28a-pelB-yin-cecB as the experimental strain, the induction conditions were systematically optimized:

Optimization of IPTG concentration: Using the BL21(DE3)/pET-28a-pelB-yin-cecB strain, activate by culturing at 37°C for 12 hours, then inoculate into 50 mL of LB medium (containing kanamycin, final concentration 50 μg/mL). When the OD₆₀₀ reaches approximately 0.6, induce in 50 mL shake flasks with IPTG at final concentrations of 0.25 mM, 0.5 mM, and 1 mM for 16 hours, respectively. Each experimental group was conducted in triplicate. The cells were lysed for protein extraction, purified, concentrated by ultrafiltration, and the protein absorbance was measured, with the mean value used for plotting.

Optimization of induction temperature: Using the E. coli BL21(DE3)/pET-28a-pelB-yin-cecB strain, culture at 37°C for 12 hours, then inoculate into 50 mL of LB medium (containing Kan, final concentration 50 μg/mL). When the OD₆₀₀ reaches approximately 0.6, add IPTG and induce at 16°C, 25°C, 30°C, and 37°C for 16 hours, respectively. Each experiment was performed in triplicate. Cells were lysed to extract protein, which was then purified and analyzed by protein gel electrophoresis to measure optical density, with the average values plotted.

July 26, 2025-July 31, 2025

Optimization of different media: Using the E. coli BL21(DE3)/pET-28a-pelB-yin-cecB strain, activate by culturing at 37°C for 12 hours, then inoculate into 50 mL of LB medium (containing Kan, final concentration 50 μg/mL) and 50 mL of TB medium (containing Kan, final concentration 50 μg/mL), with three replicates for each experiment. When the OD₆₀₀ reaches approximately 0.6, add IPTG for induction and incubate at 18°C for 16 hours. Lyse the cells to extract the protein, purify it, perform ultrafiltration protein gel to measure optical density, and use the average values to plot the data.

Optimization of different induction times: Using the E. coli BL21(DE3)/pET-28a-pelB-yin-cecB strain, activated by culturing at 37°C for 12 hours, and then inoculated into 50 mL of LB medium (containing kanamycin at a final concentration of 50 μg/mL). When the OD₆₀₀ reached approximately 0.6, IPTG was added, and the cultures were induced at 18°C for 12, 16, 20, and 24 hours. Each experiment was performed in triplicate. The cells were then lysed, protein was extracted and purified, ultrafiltration protein gel was used to measure optical density, and the average values were plotted.

August 22, 2025-August 23, 2025

Primer design: Based on the plasmid map, upstream and downstream primers targeting mutations in the pET-28a-pelB-yin-cecB plasmid were designed, namely PC-E10K-F, PC-E10K-R, PC-N15K-F, PC-N15K-R, PC-P25A-F, PC-P25A-R, PC-V29W-F, and PC-V29W-R, and synthesis was commissioned to a company.

(PC represents pET-28a-pelB-yin-cecB)

August 23, 2025

Introduce point mutations into the plasmid by PCR and remove the plasmid template using Dpn I. Gel recovery: Verify the correctness of the amplified products by agarose gel electrophoresis, and recover the DNA fragments using the FastPure Gel DNA Extraction Mini Kit.

August 24, 2025

The constructed plasmid was transformed into E. coli BL21(DE3) receptor cells, and after shaking the culture, it was spread on LB solid medium containing Kan and incubated overnight at 37°C.

August 25-August 26, 2025

It was found that PC-P25A did not grow a single colony, the rest were verified by colony PCR and sent to the company for sequencing, and the correctly sequenced strains were conserved.

August 26-August 28, 2025

PC-E10K, PC-N15K, PC-V29W were activated for 12 h, and then cultured in 50 mL of LB liquid medium (containing Kan at the final concentration of 50 μg/mL) at 37°C until OD₆₀₀≈0.6, and then added to IPTG (0.5 mM) and induced at 16°C for 20 hours. The supernatant was extracted by crushing and purified by ultrafiltration to obtain Cecropin B for the subsequent experiments.

August 29-August 31, 2025

Determination of the MIC and the spreading of the circle of inhibition of the Cecropin B against Escherichia coli and Bacillus subtilis.

August 31, 2025

Primer Design: Based on the plasmid map, upstream and downstream primers were designed for the targeted mutation in the pET-28a-pelB-yin-cecB plasmid: PC-E10K/V29W-F, PC-E10K/V29W-R, PC-E10K/N15K-F, PC-E10K/N15K-R, PC-N15K/V29W-F, and PC-N15K/V29W-R were designed based on the plasmid map and sent to the company for synthesis.

(PC denotes pET-28a-pelB-yin-cecB)

September 1, 2025-September 10, 2025

Introduce point mutations into the plasmid via PCR and remove the plasmid template using Dpn I.

Gel recovery: Verify the correctness of the amplified product through agarose gel electrophoresis and recover the DNA fragment using the FastPure Gel DNA Extraction Mini Kit.

Transform the constructed plasmid into E. coli BL21(DE3) competent cells. After shaking culture, plate the cells onto LB solid medium containing Kan and incubate overnight at 37°C.

Pick 4-7 single colonies from each plate. After verifying via colony PCR, send them to the company for sequencing. Preserve strains with confirmed sequencing results.

September 12, 2025-September 13, 2025

Activate PC-E10K/V29W, PC-N15K/V29W, and PC-E10K/N15K for 12 hours, then transferred to 50 mL LB liquid medium (containing Kan, final concentration 50 μg/mL) and cultured at 37°C until OD₆₀₀≈0.6. IPTG (0.5 mM) was added for induction at 16°C for 20 hours. The cells were lysed, proteins extracted, purified and ultrafiltration.

September 14, 2025

Determination of MIC and Bacterial Inhibition Zone Diffusion Effects for PC-E10K/V29W, PC-N15K/V29W, and PC-E10K/N15K.

August 10-August 15, 2025

Using the target strain's genomic/proteomic sequences as raw data, positive and negative sample sets were constructed in conjunction with public AMP databases. Modeling and screening: First, the protein language model (PLM) performed contextual embedding on fragments, followed by classifiers such as gradient boosting/lightweight convolutional-bidirectional gated recurrent networks to assess AMP likelihood; Subsequently, physicochemical property filtering (net positive charge, hydrophobic moment, secondary structure propensity, absence of significant toxicity/hemolytic risk, etc.) was applied to obtain high-confidence candidate monomers, designated NJT-LYY-7, NJT-LYY-4, and NJT-LYY-2.

August 16-August 20, 2025

Synthesize the peptide sequences for NJT-LYY-7, NJT-LYY-4, and NJT-LYY-2, and transform them into E. coli BL21(DE3).

August 21, 2025-August 25, 2025

Pick colonies for PCR verification, activate the correct single colonies, and inoculate into 50 mL LB broth (containing Kan, final concentration 50 μg/mL). Perform three replicates per experimental group, allowing OD₆₀₀~0.6. Add IPTG (0.5 mM) for induction, incubate at 18°C for 20 hours, then lyse and purify. Ultrafiltration of another batch is followed by lysis and lyophilization.

August 26, 2025

Testing the antibacterial efficacy and MIC of NJT-LYY-7, NJT-LYY-4, and NJT-LYY-2.

August 27-September 3, 2025

Add PelB to NJT-LYY-7, NJT-LYY-4, and NJT-LYY-2 respectively, transform into E. coli BL21(DE3), and perform colony PCR verification by streaking. Send colonies corresponding to the correct bands to the company for sequencing.

September 5, 2025-September 7, 2025

After activating the colonies corresponding to the correct results, inoculate them into 50 mL LB broth medium (containing kanamycin at a final concentration of 50 μg/mL). Perform three replicates per experimental group. When OD₆₀₀~0.6, add IPTG for induction. Incubate at 18°C for 20 hours, then lyse and purify. Ultrafiltrate another batch of lysates and freeze-dry them.

September 8, 2025-September 9, 2025

Testing the antibacterial effects and MIC of PelB-NJT-LYY-7, PelB-NJT-LYY-4, and PelB-NJT-LYY-2.

September 10, 2025

Given that the antibacterial effect of individual antimicrobial peptides is not pronounced, NJT-LYY-7, NJT-LYY-4, and NJT-LYY-2 were concatenated into a single protein chain. Computer simulations were conducted to validate the six configurations—"742," "724," "472," "427," "274," and "247"—through short-range docking and 100 ns molecular dynamics (MD) simulations. Using the RMSD of the complex backbone as the primary stability metric, the "742" combination exhibited the lowest RMSD and faster convergence to a stable plateau across multiple receptor environments and aqueous solutions. Consequently, the "742" combination was selected as the representative sequence for subsequent studies.

September 11, 2025-September 13, 2025

Construct pET-28a-pelB-LYY742 and transform it into E. coli BL21(DE3). After shaking culture, plating, and overnight static incubation, validate the construct via colony PCR. Send the correct band for sequencing at the company.

September 13, 2025-September 15, 2025

Activate the correct pET-28a-pelB-LYY742 containing colony and inoculate it into 50 mL LB broth (containing Kan, final concentration 50 μg/mL). Perform three replicates per experimental group. Upon reaching OD₆₀₀≈0.6, add IPTG (0.5 mM) for induction and incubate at 18°C for 20 hours. The lysate was purified by ultrafiltration, while another batch was lyophilized after lysis.

September 17, 2025

Run protein gel to verify band information and determine corresponding antibacterial effects and MIC.

August 10, 2025-August 25, 2025

Prepare hydrogels using materials such as polyvinyl alcohol, sodium alginate + agarose, and sodium alginate + gelatin. Validate their antibacterial efficacy using leachate, and assess their morphology and hardness.