Notebook

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.