iGEM 2025 — Team cafa-beijing

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# Notebook

This notebook chronicles not just data, but the entire journey from concept to accomplishment.

![Lab Logs hero visual](https://static.igem.wiki/teams/5920/img/t-cafabeijing-pages.webp)

[Arrangement](#overview)
[Record](#experimental)

### Arrangement

#### Summer Experimental Program

**July 1st - July 3rd:** Laboratory safety training, training on the use of basic laboratory instruments and equipment (biosafety cabinet, high-speed centrifuge, bacterial culture system, pipette, magnetic stirrer, microplate reader, inverted/upright microscope, ultrasonic cell disruptor, etc.)

**July 4th - July 7th:** Preparation of experimental materials (preparation and sterilization of LB solid/liquid culture medium, sterilization of disposable consumables, preparation of ordinary plates and antibiotic plates)

**July 8th - July 11th:** Conduct preliminary experiments, including bacterial culture, amplification, identification, competent cell preparation, and plasmid chemical transformation, to establish methodology for the formal experiments. Discuss and refine the project plan.

**July 12th - July 15th (first iteration) :** Construction, transformation, restriction enzyme analysis, expression verification, and functional verification of a target plasmid containing a recombinant fragment.

**July 16th - July 19th:** Verification of expression of the target product (ELISA or Western blot using recombinant peptide tags)

**July 20th - July 24th (second iteration):** Construction and transformation of double fusion fragment plasmid , restriction enzyme analysis, expression verification, and functional verification

**July 25th - July 30th (third iteration):** Construction and transformation of double fusion fragment plasmid , restriction enzyme analysis, expression verification, and functional verification

**August 1st - August 4th:** Amplification and culture of engineered bacteria, product purification, chemical composition identification, and sequence determination

**August 1st - August 7th (concurrent):** Synthesis of drug-loaded microneedles, loading engineered bacteria, and monitoring product release

**August 8th - August 12th:** Skin cell line recovery, expansion culture, and plating

**August 13th - August 18th:** In vivo functional validation of the product (cytotoxicity validation, melanin production inhibition validation, collagen formation promotion validation, protein expression determination, confocal microscopy of cell coverslips)

**August 15th to the end of the month:** Data collation, checking for omissions and filling gaps

### Record

#### Molecular Experiments

##### July

**Week 1 | July 1 ~ July 7**

This week focused on laboratory safety training and preparation of experimental materials, laying the foundation for subsequent experiments.

1. Laboratory Safety Training: Learned the standard operating procedures for instruments including biological safety cabinets, high-speed centrifuges, bacterial culture systems, pipettes, magnetic stirrers, microplate readers, inverted/upright microscopes, and ultrasonic cell disrupters.
2. Preparation of Experimental Materials:
   * Prepared LB solid/liquid medium and performed autoclave sterilization.
   * Prepared disposable consumables, regular plates, and antibiotic plates (e.g., containing Amp or Kan).
   * Prepared necessary reagents and buffers for experiments, such as TAE buffer, chloroform, isopropanol, etc.

**Week 2 | July 8 ~ July 11**

Conducted preliminary experiments, including bacterial culture, competent cell preparation, and plasmid transformation, to establish the methodology for formal experiments.

1. Bacterial Culture and Amplification: Revived E. coli DH5α and BL21(DE3) strains, and cultured them in LB medium in a shaker to the logarithmic phase.
2. Competent Cell Preparation: Prepared competent E. coli BL21(DE3) cells using the calcium chloride method.
3. Chemical Transformation of Plasmid: Transformed the constructed plasmid (e.g., pET-28a(+)-Melan-SD) into competent cells, plated on antibiotic plates, and preliminarily verified transformation efficiency.

**Week 3 | July 12 ~ July 15 | First Iteration**

Constructed the target plasmid containing a single recombinant fragment, and performed transformation, restriction enzyme digestion verification, and expression validation.

1. Plasmid Construction: Designed primers using SnapGene to construct the pET-28a(+)-MelanSD-His-TC-TGF plasmid.
2. Plasmid Extraction: Extracted plasmids using the alkaline lysis method.
3. Restriction Enzyme Digestion Verification: Performed double digestion on the plasmid using restriction endonucleases (e.g., XbaI and KpnI), and verified fragment size via agarose gel electrophoresis.
4. Transformation Verification: Transformed the plasmid into E. coli BL21(DE3), performed colony PCR and Sanger sequencing.

**Week 4 | July 16 ~ July 19**

Expression verification of the target product, detected via Western blot using the recombinant peptide tag.

1. Induced Expression: Used IPTG to induce the expression of the target peptide in the transformed BL21(DE3) strain.
2. Total Protein Extraction: Disrupted bacterial cells by ultrasonication, centrifuged to obtain supernatant and precipitate for SDS-PAGE analysis.

**Week 5 | July 20 ~ July 24 | Second Iteration**

Constructed the dual-fragment fusion plasmid, and performed transformation, restriction enzyme digestion verification, and functional validation.

1. Dual-Fragment Plasmid Construction: Cloned the TGF-β mimetic peptide and cell-penetrating peptide sequences into the pET-28a(+) vector.
2. Transformation and Verification: Transformed the constructed plasmid into E. coli DH5α and BL21(DE3), performed colony PCR and restriction enzyme digestion verification.
3. Expression Verification: Verified the expression of the fusion peptide via SDS-PAGE and Ponceau S staining.

**Week 6 | July 25 ~ July 30 | Third Iteration**

Further optimized the dual-fragment fusion plasmid and performed functional validation.

1. Plasmid Reconfiguration: Adjusted vector design based on previous iteration results, such as replacing the promoter or tag sequences.
2. Transformation and Expression: Performed re-transformation and induced expression, verifying expression via SDS-PAGE and immunofluorescence.
3. Preliminary Functional Verification: Used the CCK-8 assay to detect the effect of peptide sequences on the viability of L-929 cells.

#### August

**Week 7 | Aug 1 ~ Aug 4**

Large-scale culture of engineered bacteria, product purification, and chemical identification.

1. Large-Scale Culture: Scaled up the culture of successfully transformed BL21(DE3) engineered bacteria.
2. Product Purification: Purified the His-tagged fusion peptide using Ni-NTA affinity chromatography.
3. Mass Spectrometry Identification: Verified the correct peptide sequence using MALDI-TOF mass spectrometry.

**Week 8 | Aug 5 ~ Aug 7 | Conducted Concurrently**

Synthesis of drug-loaded microneedles and loading with engineered bacteria.

1. Microneedle Preparation: Prepared biodegradable microneedle arrays using polymer materials.
2. Bacterial Loading: Loaded the engineered bacterial suspension into the microneedles, optimizing loading efficiency.
3. Release Monitoring: Monitored the release kinetics of the peptide in a simulated skin environment.

**Week 9 | Aug 8 ~ Aug 12**

Revival, expansion culture, and plating of skin cell lines.

1. Cell Revival: Revived the L-929 mouse fibroblast cell line.
2. Expansion Culture: Expanded cells in DMEM medium for subsequent toxicity and functional verification.
3. Plating Preparation: Seeded cells into 96-well or 24-well plates for experiments such as CCK-8 and immunofluorescence.

**Week 10 | Aug 13 ~ Aug 18**

In vivo functional verification of the product.

1. Cytotoxicity Verification: Used the CCK-8 assay to detect the effect of different concentrations of peptide solutions on the viability of L-929 cells.
2. Melanin Production Inhibition Verification: Detected TRP1 expression via immunofluorescence.
3. Protein Expression Measurement: Analyzed changes in TRP1 expression in MSH knockdown cell lines using Western blot.
4. Confocal Microscopy Observation: Performed DAPI and FITC staining on cell爬片 to observe the effect of the peptide on cell morphology and protein localization.

**Week 11 | Aug 15 ~ Aug 31**

Data organization and filling in gaps.

1. Organized all experimental data, including SDS-PAGE, Western blot, CCK-8, immunofluorescence images, etc.
2. Statistical Analysis: Performed data significance analysis using GraphPad Prism.
3. Supplementary Experiments: Repeated key experiments or optimized conditions based on preliminary results.

#### Validation Experiments

##### July

**Week 4 | July 16 ~ July 19**

Conducted cell compatibility tests to verify the effect of peptide sequences on cell viability.

1. Preparation of Peptide Solutions: Diluted the purified peptide sequences in concentration gradients (300, 100, 33.3, 11.1, 3.7, 1.2, 0.4, 0.14 μM).
2. CCK-8 Detection: Co-cultured L-929 cells with peptide solutions, added CCK-8 reagent, and measured the absorbance at 450 nm.
3. Data Analysis: Calculated cell survival rates to confirm that the peptide sequences showed no significant toxicity.

**Week 5 | July 20 ~ July 24**

Conducted melanin inhibition function verification to evaluate the effect of peptides on TRP1 expression.

1. Immunofluorescence Staining: Fixed L-929 cells, performed staining using an anti-TRP antibody and an FITC-labeled secondary antibody.
2. Fluorescence Microscopy Observation: Detected TRP1 fluorescence intensity and quantified expression levels.
3. Result Analysis: Peptide sequences EERVEEGRR, EAIVRYFAG, and EEIVEYFAG all showed downregulation of TRP1 expression.

#### August

**Week 10 | Aug 13 ~ Aug 18**

Conducted suicide switch function verification, dynamically observing the bacterial lysis effect.

1. Time-kill Curve: Sampled at different time points after adding arabinose, plated and counted colonies.
2. Live/Dead Bacterial Staining: Used Calcein-AM and PI staining, observed bacterial survival via fluorescence microscopy.
3. Data Analysis: Plotted the bactericidal curve, confirming that the suicide switch could effectively induce bacterial death.

**Week 11 | Aug 19 ~ Aug 31**

Conducted verification of the MSH knockdown cell line and analysis of TRP1 expression.

1. siRNA Transfection: Designed MSH-specific siRNA and transfected L-929 cells.
2. RT-qPCR Detection: Extracted total RNA, synthesized cDNA, and performed real-time quantitative PCR to analyze MSH mRNA expression.
3. Western Blot Verification: Used the total membrane stripping and re-probing method to detect changes in TRP1 protein expression.

Reference: Velimirovic M, Zanetti LC, Shen MW, Fife JD, Lin L, Cha M, Akinci E, Barnum D, Yu T, Sherwood RI. Peptide fusion improves prime editing efficiency. Nat Commun. 2022 Jun 18;13(1):3512. doi: 10.1038/s41467-022-31270-y. PMID: 35717416; PMCID: PMC9206660.et al.

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# Engineering

Engineering is a cyclical process, where each attempt brings new insights for the next step.

![Feasibility hero visual (microneedle & collagen concept)](https://static.igem.wiki/teams/5920/img/t-cafabeijing-pages.webp)

### Overview

Aiming to, we designed four experiment cycles under the guidance of DBSL rules. The goals of the four groups of engineering modifications are:

1. Verify that the selected strain can grow normally in experimental environments we have constructed.
2. Use protein de novo design tools semi-rationalally design the mutation library who’s prototype, then screened out targeted MSH-α analogues.
3. Introduce and express the TGF protein.
4. Build controlled bacterial clearance.

### Cycle 1 — Construction & Band Verification

C1

![Design icon](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng-icon4.webp)Design
![Build icon](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng-icon3.webp)Build
![Test icon](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng-icon2.webp)Test
![Learn icon](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng-icon1.webp)Learn

#### Design

In the first engineering design cycle, our goal is to test whether the engineered bacterial strain selected in the experimental design could grow normally under the experimental conditions.The main objective of this stage is to establish a standardized and highly successful experimental foundation for the subsequent experiments.We have selected the following two strains：DH5-α and Escherichia coli BL21(DE3). DH5α is a mutant strain of Escherichia coli, mainly characterized by its lack of immunity to exogenous DNA. Compared to normal strains, it lacks certain immune mechanisms，can be used to create competent cells for genetic engineering.The Escherichia coli BL21(DE3) strain is a type of Escherichia coli expression strain, using T7 RNA polymerase as the expression system. It can efficiently express genes cloned into expression vectors containing the phage T7 promoter. The λ phage DE3 region contains the T7 phage RNA polymerase gene, and this region is integrated onto the chromosome of BL21.

#### Build

We used LB solid medium to cultivate DH5-α and Escherichia coli BL21(DE3), observe its growth condition and record. Introduce the plasmid into Escherichia coli BL21(DE3) and DH5-α, then test the transformation process through agarose gel electrophoresis.

#### Test

![Fig.1 Bacterial plate of Escherichia coli BL21(DE3) (left) and DH5-α(right)](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng1.webp)

Fig.1 Bacterial plate of Escherichia coli BL21(DE3) (left) and DH5-α(right)

![Fig.2 Result of agarose gel electrophoresis](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng2.webp)

Fig.2 Result of agarose gel electrophoresis

#### Learn

By observing its growth rate and colony morphology, we found that this strain can grow normally in the environment we have constructed.This ensured the smooth progress of the subsequent experiments, and through repeated experiments, the influence of the cultivation environment factors on the experimental results was eliminated.

### Cycle 2 — Expression & Preliminary Readout

C2

![Design icon](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng-icon4.webp)Design
![Build icon](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng-icon3.webp)Build
![Test icon](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng-icon2.webp)Test
![Learn icon](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng-icon1.webp)Learn

#### Design

We use a series of protein de novo design tools driven by artificial intelligence, semi-rationalally design the mutation library, who’s prototype bases on MSH-α. Then screened out targeted MSH-α analogues use affinity and biocompatibility as indicators. Finally, the expression systems were introduced into Escherichia coli BL21(DE3) using plasmid pET-28a(+)\_Melan-SD.

![Fig.3 Plasmid pET-28a(+)_Melan-SD](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng003.webp)

Fig.3 Plasmid pET-28a(+)\_Melan-SD

For more detailed information, please refer to ***[Model](https://2025.igem.wiki/cafa-beijing/model)***.

#### Build

![Fig.4 Result through RFdiffusion](https://static.igem.wiki/teams/5920/img/t-cafabeijing-model2.webp)

Fig.4 Result through RFdiffusion

![Fig.5 Result through ProteinMPNN](https://static.igem.wiki/teams/5920/img/t-cafabeijing-model3.webp)

Fig.5 Result through ProteinMPNN

![Fig.6  Result through Autodock Vina](https://static.igem.wiki/teams/5920/img/t-cafabeijing-model4.webp)

Fig.6 Result through Autodock Vina

#### Test

![Fig.7 Cell Viability Under Co-Cultivation with Different Peptide Sequences](https://static.igem.wiki/teams/5920/img/t-cafabeijing-model9.webp)

Fig.7 Cell Viability Under Co-Cultivation with Different Peptide Sequences

![Fig.8 Expression of TRP1 Protein in Cells Under Different Peptide Intervention Conditions](https://static.igem.wiki/teams/5920/img/t-cafabeijing-model10.webp)

Fig.8 Expression of TRP1 Protein in Cells Under Different Peptide Intervention Conditions

For more detailed information, please refer to ***[Results](https://2025.igem.wiki/cafa-beijing/results)***.

#### Learn

The short peptides we designed have been proven to have the function of inhibiting melanin production.

### Cycle 3 — Condition Optimization & Quantification

C3

![Design icon](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng-icon4.webp)Design
![Build icon](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng-icon3.webp)Build
![Test icon](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng-icon2.webp)Test
![Learn icon](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng-icon1.webp)Learn

#### Design

Using plasmids, we introduced the TGF-β protein expression system into Escherichia coli, enabling the expression product to simultaneously achieve whitening and anti-aging effects. TGF-β is a type of cytokine that can induce the expression of the c-sis gene in fibroblasts. Define an optimization matrix (media, induction strength, expression temperature; optional promoter/RBS swaps). Pre-register quantitative metrics: live/dead ratio, per-channel intensity, growth metrics.

#### Build

We used a His-tag enzyme-linked immunosorbent assay (ELISA) to detect the expression of TGF-6xHis, which can indicating the expression and extracellular secretion of TGF-β.

#### Test

![Fig.9 Result of His-tag enzyme-linked immunosorbent assay (ELISA)](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng9.webp)

Fig.9 Result of His-tag enzyme-linked immunosorbent assay (ELISA)

![Fig.10 Result of the Type I collagen (COL-1) expression](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng10.webp)

Fig.10 Result of the Type I collagen (COL-1) expression

#### Learn

The results showed that compared to the blank control group, E. coli culture supernatant, and BL21 (DE3) culture supernatant, the His signal in the supernatant of the engineered bacteria significantly increased, indicating the successful expression and extracellular secretion of TGF-β. And the supernatants from engineered bacteria expressing only the melanotropin peptide, as well as those co-expressing the melanotropin peptide and TGF, both upregulated collagen expression in L-929 cells. During this cycle, we successfully obtained the dual-effect product as required.

### Cycle 4 — Condition Optimization & Quantification

C4

![Design icon](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng-icon4.webp)Design
![Build icon](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng-icon3.webp)Build
![Test icon](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng-icon2.webp)Test
![Learn icon](https://static.igem.wiki/teams/5920/img/t-cafabeijing-eng-icon1.webp)Learn

#### Design

To achieve controlled death of engineered bacteria, we constructed a toxin-antitoxin plasmid regulated by an arabinose operon. Referred to the suicide switch constructed by CAFA\_China2022, SZ-SHD, we combined T4 endolysin(Part:BBa\_K112806) and T4 holin(Part:BBa\_K112805), created a composite suicide switch plasmid. then made adjustments based on BL21(DE3), and other plasmids that were introduced simultaneously. The T4 endolysin is lysozyme from enterobacteria phage T4 degrades peptidoglycan layer. The T4 holin is holins from T4 bacteriophage assemble together to form pores on inner membrane of bacteria allowing lysozyme to reach periplasm and degrade peptidoglycan layer.The combination of the two can mutually enhance each other.

![Fig.11 the pBAD30-T4-lysis plasmid](https://static.igem.wiki/teams/5920/img/t-cafabeijing-parts1.webp)

Fig.11 the pBAD30-T4-lysis plasmid

#### Build

We constructed the pBAD30-T4-lysis plasmid and transformed it into E. coli BL21 (DE3).

#### Test

We used live/dead bacterial fluorescence staining and colony count-based bactericidal curve measurement to verify whether the suicide switch system can control bacterial apoptosis. The results showed significant death signals in bacteria 24 hours after the addition of arabinose. Colony count results indicated that the number of colonies decreased to zero after 24 hours.

![Fig.12 Live/dead bacterial staining indicates that red fluorescence signals represent dead bacteria](https://static.igem.wiki/teams/5920/img/t-cafabeijing-parts2.webp)

Fig.12 Live/dead bacterial staining indicates that red fluorescence signals represent dead bacteria

![Fig.13 Time-series bactericidal curve](https://static.igem.wiki/teams/5920/img/t-cafabeijing-parts3.webp)

Fig.13 Time-series bactericidal curve

#### Learn

The suicide plasmid can function in E. coli BL21 (DE3) properly.

### Basic Part

We designed multiple short peptide sequences using a computer-aided approachand selected three sequences that demonstrated relatively high stability in actualexperiments, which have been submitted as Basic Parts.
They are
[BBa\_25682NRB](https://registry.igem.org/parts/bba-25682nrb),
[BBa\_25NPAQ79](https://registry.igem.org/parts/bba-25npaq79),
and
[BBa\_257Q5CBQ](https://registry.igem.org/parts/bba-257q5cbq)<

**Functional Description:** Encoded under the lPTG-inducible T7 promoter, this melanin-inhibitory peptide functions as a competitive peptide analog targeting the a-melanocyte-stimulating hormone receptor (a-MSH). lt effectively blocks melanin synthesis while simultaneously promoting collagen production.

Reference: Velimirovic M, Zanetti LC, Shen MW, Fife JD, Lin L, Cha M, Akinci E, Barnum D, Yu T, Sherwood RI. Peptide fusion improves prime editing efficiency. Nat Commun. 2022 Jun 18;13(1):3512. doi: 10.1038/s41467-022-31270-y. PMID: 35717416; PMCID: PMC9206660.et al.

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# Results

Our data demonstrate not only the soundness of the design but also the engineered bacteria’s stable expression and self-regulation.

![Results hero visual](https://static.igem.wiki/teams/5920/img/t-cafabeijing-pages.webp)

[1 Peptide Sequence Prediction](#overview)
[2 Peptide Function Validation](#experimental)
[3 Expression System](#expression-system)
[4 The Functional Validation of Recombinant Bacteria](#functional-bacteria)
[5 Controlled bacterial clearance](#bacterial-clearance)

### 1 Peptide Sequence Prediction

**1.1. Protein De Novo Design**

We use a series of protein de novo design tools driven by artificial intelligence, semi-rationalally design the mutation library, who’s prototype bases on MSH-α.

![Fig.1 Result through RFdiffusion](https://static.igem.wiki/teams/5920/img/t-cafabeijing-model2.webp)

Fig.1 Result through RFdiffusion

We use a series of protein de novo design tools driven by artificial intelligence, semi-rationalally design the mutation library, who’s prototype bases on MSH-α.

![Fig.2 Result through ProteinMPNN](https://static.igem.wiki/teams/5920/img/t-cafabeijing-model3.webp)

Fig.2 Result through ProteinMPNN

We use a series of protein de novo design tools driven by artificial intelligence, semi-rationalally design the mutation library, who’s prototype bases on MSH-α.

![Fig.3 Result through Autodock Vina](https://static.igem.wiki/teams/5920/img/t-cafabeijing-model4.webp)

Fig.3 Result through Autodock Vina

**1.2. Multiple Sequence Alignment**

We integrated all sequences into a global multiple sequence alignment. During this process, gaps were permitted to maximize similarity among sequences, while gap opening penalties and gap extension penalties were imposed to maintain biological plausibility.

![Fig.2 Peptide Sequence Prediction Results](https://static.igem.wiki/teams/5920/img/t-cafabeijing-result1-4.webp)

Fig.2 Peptide Sequence Prediction Results

### 2 Peptide Function Validation

**2.1 Cell Compatibility Test**

To verify that the designed peptide sequences do not pose potential harm to tissue cells, we selected the top 8 purified peptide sequences based on their binding energy rankings. Different concentrations of peptide solutions were added to the culture medium of mouse fibroblasts (L-929) and co-cultured with the cells. The cell viability of the different groups was subsequently tested using the CCK-8 detection method. The CCK-8 assay is simple yet accurate for analyzing cell proliferation and cytotoxicity.

The CCK-8 reagent contains the water-soluble tetrazolium salt WST-8 [Chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt], which is reduced by dehydrogenases in cells under the action of the electron carrier 1-methoxy-5-methylphenazinium methyl sulfate (1-Methoxy PMS) to produce a highly water-soluble yellow formazan product. The amount of formazan produced is directly proportional to the number of living cells. This characteristic allows direct analysis of cell proliferation and toxicity.

![Fig.1 Cell Viability Under Co-Cultivation with Different Peptide Sequences](https://static.igem.wiki/teams/5920/img/t-cafabeijing-result2-1.webp)

Fig.1 Cell Viability Under Co-Cultivation with Different Peptide Sequences

**2.2. Melanin Inhibition Function Validation**

To verify whether the designed peptide sequences can inhibit melanin production, we selected tyrosinase-related protein 1 (TRP1), which possesses 5,6-dihydroxyindole-2-carboxylic acid oxidase activity and plays an important role in the downstream pathways of melanin synthesis. TRP1 can also be activated through the MSH receptor and its downstream signaling pathways.

To assess the effect of peptide intervention on melanin synthesis, we used immunofluorescence assays with an FITC-labeled green fluorescent secondary antibody to evaluate TRP1 expression in cells. The results demonstrated that the peptides ***EERVEEGRR***, ***EAIVRYFAG***, and ***EEIVEYFAG*** all reduced TRP1 expression. The fluorescence intensity of TRP1 was measured as a percentage relative to the control group, showing values of ***62.1*** ± ***3.5%***, ***75.4*** ± ***4.9%***, and ***72.3*** ± ***2.8%***, respectively. This suggests these peptides effectively downregulate TRP1 expression and may consequently inhibit melanin synthesis.

![Fig.2 Expression of TRP1 Protein in Cells Under Different Peptide Intervention Conditions](https://static.igem.wiki/teams/5920/img/t-cafabeijing-result2-2.webp)

Fig.2 Expression of TRP1 Protein in Cells Under Different Peptide Intervention Conditions

### 3 Expression System

**3.1. Plasmid Construction and Amplification**

To express the required peptide sequence in engineered bacteria and apply specific modifications to its N- and C-terminals, we constructed the expression system ***pET-28A(+)-MelanSD-His-TC-TGF*** using the ***pET-28A(+)*** plasmid backbone.

We first transformed the plasmid into ***Escherichia coli DH5α*** using an improved calcium rubidium method to amplify its quantity. Subsequently, the plasmid was extracted and retransformed into ***Escherichia coli BL21(DE3)*** to construct an IPTG-inducible peptide expression system.

The results of agarose gel electrophoresis showed that the amplified bands from the transformed **DH5α E. coli** were consistent with the theoretical plasmid size, confirming that the ***pET-28A(+)-MelanSD-His-TC-TGF*** plasmid was successfully transformed into the bacteria and efficiently amplified.

![Fig.1 The agarose gel electrophoresis results of the plasmid extracted from E. coli DH5α colonies matched the pET-28A(+)-MelanSD-His-TC-TGF plasmid](https://static.igem.wiki/teams/5920/img/t-cafabeijing-result3-1.webp)

Fig.1 The agarose gel electrophoresis results of the plasmid extracted from E. coli DH5α colonies matched the pET-28A(+)-MelanSD-His-TC-TGF plasmid

**3.2 Verification of Target Peptide Expression**

To detect whether the transformed BL21 (DE3) engineered bacteria could successfully express the target peptide under IPTG induction, we analyzed the total bacterial protein using SDS-PAGE and ***Ponceau S staining*** staining methods. The results showed intensified staining in the lanes corresponding to the predicted molecular weight of the Melan fusion peptide (13-16 kDa). In contrast, no intensified staining was observed at this position in uninduced, non-plasmid-transformed BL21 (DE3), or DH5α bacteria. This demonstrated that the engineered bacteria successfully expressed the protein with the expected molecular weight.

![Fig.2 SDS-PAGE image](https://static.igem.wiki/teams/5920/img/t-cafabeijing-result3-2.webp)

Fig.2 SDS-PAGE image

### 4 The Functional Validation of Recombinant Bacteria

**4.1. MSH knockdown cell line**

To verify whether the expression product of the recombinant bacteria can inhibit melanin production by targeting the MSH receptor and its downstream signaling pathways, we first constructed an MSH gene knockdown L-929 cell line. Specifically, we designed an siRNA that interacts with the mRNA corresponding to MSH and used a transfection reagent (Lip2000) to mix with the siRNA for cell transfection. Subsequently, total RNA was extracted from the cells, and RT-qPCR was performed to detect the mRNA expression of MSH.

The results showed that compared to the control group, the MSH expression levels in both the NC group and the siRNA group were reduced, indicating the successful establishment of the MSH knockdown cell line.

![Fig.1 Quantitative analysis of PCR amplification curves](https://static.igem.wiki/teams/5920/img/t-cafabeijing-result4-1.webp)

Fig.1 Quantitative analysis of PCR amplification curves

**4.2. Verification of TRP1 expression**

We used the whole membrane stripping and re-probing method in Western blot to simultaneously detect the expression of the reference protein and TRP-1 protein. The results showed that TRP-1 expression decreased in cells co-cultured with the filtered product of the recombinant bacterial culture supernatant. After knocking down MSH, TRP-1 expression increased, indicating that the filtered product can inhibit TRP-1 expression and is regulated by MSH.

![Fig.2 Western-blot](https://static.igem.wiki/teams/5920/img/t-cafabeijing-result4-2.webp)

Fig.2 Western-blot

### 5 Controlled bacterial clearance

**5.1 Construction and transformation of suicide plasmid**

To achieve controlled death of engineered bacteria, we constructed a toxin-antitoxin module regulated by an arabinose operon. When the engineered bacteria accidentally leak or reach conditions requiring termination, arabinose can replace glucose as an energy source and interact with the arabinose operon to initiate suicide gene transcription. We constructed the ***pBAD30-T4-lysis*** plasmid and transformed it into E. coli BL21 (DE3).

![Fig.1 BL21(DE3) bacteria, BL21(DE3) bacteria transformed with the pET-28A(+)-MelanSD-His-TC-TGF plasmid, and bacteria transformed with both the pET-28A(+)-MelanSD-His-TC-TGF and pBAD30-T4-lysis plasmids.](https://static.igem.wiki/teams/5920/img/t-cafabeijing-result5-1.webp)

Fig.1 BL21(DE3) bacteria, BL21(DE3) bacteria transformed with the ***pET-28A(+)-MelanSD-His-TC-TGF*** plasmid, and bacteria transformed with both the ***pET-28A(+)-MelanSD-His-TC-TGF*** and ***pBAD30-T4-lysis*** plasmids.

**5.2 Verification of Functionality**

We used live/dead bacterial fluorescence staining and colony count-based bactericidal curve measurement to verify whether the suicide switch system can control bacterial apoptosis. The results showed significant death signals in bacteria 24 hours after the addition of arabinose. Colony count results indicated that the number of colonies decreased to zero after 24 hours.

![Fig.2 Live/dead bacterial staining indicates that red fluorescence signals represent dead bacteria.](https://static.igem.wiki/teams/5920/img/t-cafabeijing-result5-2.webp)

Fig.2 Live/dead bacterial staining indicates that red fluorescence signals represent dead bacteria.

![Fig.3 Time-series bactericidal curve](https://static.igem.wiki/teams/5920/img/t-cafabeijing-result5-3.webp)

Fig.3 Time-series bactericidal curve

Reference: Velimirovic M, Zanetti LC, Shen MW, Fife JD, Lin L, Cha M, Akinci E, Barnum D, Yu T, Sherwood RI. Peptide fusion improves prime editing efficiency. Nat Commun. 2022 Jun 18;13(1):3512. doi: 10.1038/s41467-022-31270-y. PMID: 35717416; PMCID: PMC9206660.et al.

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