iGEM 2025 — Team fhs-nb-zj-china

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## Overview

Many teammates **collaborated to complete** our experiments.

pET-CH1 plasmid construction was carried out by Chenjiong Wan, Yixiang Sun, Jinyao Chu, Yingjie Sun and Ye Zhu, while pET-F4 plasmid construction was carried out by Sibo Hu, Ziqian Wu, Jiaye Li, Xinxin Li, Ni Liu and Yuxuan Ma.

### Literature Study and dsRNAs Design

### June 12 - July 4, 2025

**Aim:**

* To search for dsRNA design and prediction literatures and tools.
* Recent researches on *Lasiodiplodia theobromae*.
* Double-stranded RNA (dsRNA) for RNA interference (RNAi) targeting *Chitin Synthase* (predicted) in *Lasiodiplodia theobromae*.

**Protocols:**

* Both international and national databases were used.
* [Oligowalk](https://rna.urmc.rochester.edu/cgi-bin/server_exe/oligowalk/oligowalk_form.cgi) from Mathews group was used for siRNA design.

**Results:**

* Partial mRNA sequence of Chitin Synthase in *Lasiodiplodia theobromae*  was downloaded from [GenBank](https://www.ncbi.nlm.nih.gov/nuccore/XM_035509731.1).
* Nucleitide sequence of sGFP was provided by PLD Technology (the supplier of cell-free protein expression kit we used last year).
* Three dsRNA fragments, named F1-3 targeting Chitin Synthase in *Lasiodiplodia theobromae* and one dsRNA, named F4 fragment targeting *superfold GFP* were designed.

**Table 1. Four dsRNA fragments sequences.**

| Name | Sequence |
| --- | --- |
| F1 | CCGGAATTCCGGATGTCGAGTACAAGATGAGTAATTCTCGAGTGTTACTCATCTTGTACTCGATAGTAGCCCAAGCTTGGG |
| F2 | CCGGAATTCCGGATGCCGACGATGCTGATTTGAATTCTCGAGTGTTCAAATCAGCATCGTCGGTAGTAGCCCAAGCTTGGG |
| F3 | CCGGAATTCCGGATGGCTCACGTTTACGAGTACATTCTCGAGCGTGTACTCGTAAACGTGAGCTAGTAGCCCAAGCTTGGG |
| F4 | CCGGAATTCCGGATGGCATTAAAGCGAATTTTAATTCTCGAGTTTTAAAATTCGCTTTAATGCTAGTAGCCCAAGCTTGGG |

* For safety reasons, we cannot culture the pathogenic fungi that cause peach gummosis, and thus cannot verify whether the designed dsRNAs can interfere with their growth. Instead, if we can develop dsRNA that interferes with the synthesis of sGFP, we will be able to determine the success of the experiment with the naked eye. So cloning experiment was only carried out on F4 fragment. Key structural features of F4 fragment:
  + **Restriction Enzyme Recognition Sites:**
    - An *Eco*RI restriction site (GAATTC) is present at the 5' end (sequence position: 5-10).
    - A *Hin*dIII restriction site (AAGCTT) is present at the 3' end (sequence position: 64-69).
  + **Sequence Composition**:
    - Total length: 73 base pairs (bp).
    - Protective bases (CCG at the 5' end and GGG at the 3' end) are attached to both ends to enhance restriction enzyme digestion efficiency.

### Gene Fragments and Primers Synthesis

### June 26 - July 18, 2025

**Notes:**

* Nucleitide sequence of *Prunus persica chitinase*, named CH1 was downloaded from [KEGG](https://www.genome.jp/dbget-bin/www_bget?pper:18792824).
* *CH1* gene fragments was synthesized free of charge by GenScript China.

**Table 2. Primers and synthesized DNA fragment used in this project**.

| Name | Sequence |
| --- | --- |
| T7\_P | TAATACGACTCACTATAGGG |
| T7\_T | TGCTAGTTATTGCTCAGCGG |
| CH1\_F1 | ATGGGTCGCGGATCCGAATTCATGGCTAGCTATAGACTACTAAGCCTCA |
| CH1\_R1 | CTCGAGTGCGGCCGCAAGCTTCTAGCAGGAGAGATTAGTGCCAGG |
| F4 | CCGGAATTCCGGATGGCATTAAAGCGAATTTTAATTCTCGAGTTTTAAAATTCGCTTTAATGCTAGTAGCCCAAGCTTGGG |

### GFP plasmid and pET-28a(+) plasmid amplification

### July 14 - 22, 2025

**Aim:**

* Amplification of commercial GFP plasmid DNA (PLD Technology) by BL21(DE3) and pET-28a(+) plasmid DNA (Yeasen Biotechnology) by DH5α.
* To test the activities of the DH5α super competent cells purchased last year.

**Protocols:**

* Transformation by using BL21(DE3) and DH5α super competent cells (Beyotime Biotechnology)
* Plasmid DNA isolation by Plasmid Miniprep kit (Beyotime Biotechnology)
* dsDNA concentration determination by Qubit dsDNA High Sensitivity kit (Thermo fisher) and Fluorometer Gen5 (Leader Technology)
* *Sac*I (Beyotime Biotechnology) digestion for validation.

**Results:**

* DNA concentrations of GFP vector and pET-28a(+) were 17 ng/μl and 37 ng/μl respectively.
* The electrophoresis was conducted under 100 V for 30 minutes in TAE buffer.
![Fig. 1](https://static.igem.wiki/teams/5595/msds/gelphoto1.webp)

Fig. 1 DNA agarose gel results

1: GFP plasmid (Extracted on 21st July 2025, target: 2,510 bp);2: *Sac* digestion of the GFP plasmid (Digested on 22nd July 2025, target: 2,510 bp); 3: pET-28a(+) plasmid (Extracted on 21st July 2025, target: 5,369 bp); 4: *Sac* digestion of the pET-28a(+) plasmid (Digested on 22nd July 2025, target: 5,369 bp); 5,6: CH1 PCR products (Amplified on 22nd July 2025, target: 732 bp); 7: non-template control for 5&6; 8: non-template control for 10; 9: non-template control for 11; 10: CH1 PCR products (Amplified on 17th July 2025, target: 732 bp); 11: 038 PCR product (Amplified on 17th July 2025, target: 1,956 bp); 12: 100-2000 bp DNA ladder.

### Construction of A Recombinant pET-CH1 Plasmid

### July 23 - August 3, 2025

**Aim:**

* To generate a linearized and modified pET-28a(+) vector for subsequent cloning applications.
* To construct a recombinant pET-CH1 plasmid for protein expression and functional analysis.
* Digestion, extraction, cloning and transformation enabled construction and propagation of the target recombinant vector.

**Protocols:**

* Double enzyme digestion pET-28a(+) vector with *Eco*RI & *Hin*dIII (double digestion).
* Cloning of the target fragment into linearized pET-28a(+) , screening by colony PCR, and the recombinant plasmid was extracted.
* Purification of double-digested pET-28a(+) vector via gel extraction.
* Transformation of the assembled pET-CH1 construct into BL21(DE3) super competent cells for propagation and expression.

**Results:**

* pET-CH1 plasmid was successfully constructed.
![Fig. 2](https://static.igem.wiki/teams/5595/notebook/ch1.webp)

Fig. 2 *E. coli* plaque results

![Fig. 3](https://static.igem.wiki/teams/5595/notebook/e-coli-colony-pcr-ch1.webp)

Fig. 3 Colony PCR resutls of pET-CH1.

* Sanger sequencing results confirmed our construct was right.

### Construction of A Recombinant pET-F4 Plasmid

### August 15 - 18, 2025

**Aim:**

* To construct and isolate a recombinant plasmid for expression of anti-GFP dsRNA in *E. coli.*

**Protocols:**

* Double restriction digestion of the F4 fragment and pET-28a(+) vector, followed by ligation with T4 DNA ligase
* Transformation of the ligation product into *E. coli* DH5α super competent cells (Beyotime Biotechnology) for plasmid propagation
* Plasmid DNA extraction from positive clones.

**Results:**

![Fig. 4](https://static.igem.wiki/teams/5595/notebook/f4.webp)

Fig. 4 Double digested of F4 fragment

1,2: The F4 fragment after double digestion; 3: 100-600 bp DNA ladder.

![Fig. 5](https://static.igem.wiki/teams/5595/notebook/f4-plate.webp)

Fig. 5 pET-F4 *E. coli* plaque results

![Fig. 6](https://static.igem.wiki/teams/5595/notebook/pet-28a-f4-pcr250817.webp)

Fig. 6 Colony PCR resutls of pET-F4.

1: 100-600 bp DNA ladder; 2,3,4: LJY 1~3; 5,6,7: MYX 1~3; 8,9: LXX 1~2; 10: WZQ; 11: HSB; 12: CK; 13: 100-2000 bp DNA ladder.

* Sanger sequencing results indicated something wrong with our constructs. Only part of F4 fragment was inserted in to the vector, which may due to the secondary structure of the dsRNA.

  ![Fig. 7](https://static.igem.wiki/teams/5595/notebook/f4mis.avif)

### Induced Expression and Validation of pET-CH1 Plasmid

### August 23 - 31, 2025

**Aim:**

* Induced expression of pET-CH1

**Protocols:**

* Expression of pET-CH1 by IPTG (0.5 mM). (Beyotime Biotechnology)
* Total protein extration by kit. (Beyotime Biotechnology)
* Validation of CH1 Expression by SDS-PAGE and Coomassie Staining. (Beyotime Biotechnology)

**Results:**

We failed to determine which band corresponds to our target protein.

![Fig. 8](https://static.igem.wiki/teams/5595/notebook/total-protein-sds-page.webp)

Fig. 9 SDS-PAGE gel results of total protein samples with coomassie staining.

1, 10: Denatured Protein Ladder (14.4-116kD); 2: Empty vector; 3: CH1-1 0 h; 4: CH1-3 0 h; 5: CH1-3 2 h; 6: CH1-3 4.5 h; 7,8: GFP plasmid; 9: CH1-1 4.5 h

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## Design-Build-Test-Learn Cycles

#### Three Wet-lab-based DBTL cycles

pET-CH1

pET-F4

SDS-PAGE

Design

Build

Learn

Test

DBTL

### Design

To express peach chitinase at a high level and inducible, we chose the pET-28a(+) vector, which was driven by the T7 promoter.

### Build

1. Nucleotide sequence of chitinase *Prunus persica* (peach), named CH1 in this study was downloaded from [KEGG](https://www.genome.jp/dbget-bin/www_bget?pper:18792824).
2. *PpCH1* gene fragments was synthesized free of charge by GenScript China.
3. Amplification of pET-28a(+) plasmid DNA by DH5α, which was linearized for subsequent cloning applications.
4. A recombinant pET-CH1 plasmid was constructed and confirmed by sequencing.

### Learn

We tried twice, but failed to determine which band corresponds to our target protein. The total protein extraction process should be optimized.

### Test

A recombinant pET-CH1 plasmid was used for protein expression analysis.

Design

Build

Learn

Test

DBTL

### Design

To express specific dsRNA: F4 fragment for the purpose of knocking down sGFP expression.

1. The nucleotide sequence of superfold GFP (sGFP) was provided by PLD Technology.
2. [Oligowalk](https://rna.urmc.rochester.edu/cgi-bin/server_exe/oligowalk/oligowalk_form.cgi) from Mathews group was used for siRNA design.
3. A double-stranded RNA (dsRNA) fragment targeting sGFP (F4 fragment) was designed with restriction sites.

### Build

1. F4 fragment was synthesized by GenScript.
2. Double restriction digestion of F4 fragment and pET-28a(+) vector.
3. Ligation with T4 DNA ligase and transformation into *E. coli* DH5α.
4. Plasmid extraction and confirmation by sequencing.

### Learn

The short F4 fragment and secondary structure interference with cloning are issues that require testing of alternative cloning strategies.

### Test

Sanger sequencing results indicated **incomplete insertion of F4 fragment** into the vector.

Design

Build

Learn

Test

DBTL

### Design

To observe protein by performing SDS-PAGE safely in a high school laboratory with precast PAGE Gels.

### Build

Total protein extraction from *E. Coli*.

Perform SDS-PAGE.

### Learn

Although we did not obtain the expected clear bands for robust expression, the established experimental workflow demonstrates the feasibility of our cloning strategy, confirming that our system can support protein expression with further refinement.

Faint bands also indicated partial expression, and further optimization of the protein extraction protocol, induction time, and IPTG concentration is needed to improve yield.

### Test

We failed to determine which band corresponds to our target protein.

## Engineering Success

#### Key Achievements:

* An IPTG-inducible expression system for the predicted chitinase from *Prunus persica* (peach) (the CDS part: [BBa\_25CSQLVI](https://registry.igem.org/parts/bba-25csqlvi)) was successfully constructed using the T7-driven pET-28a vector ([BBa\_25YSLUTM](https://registry.igem.org/parts/bba-25yslutm)).
* Two methods were employed for plasmid construction: homologous recombination and double restriction digestion with T4 DNA ligase ligation.
* The process of performing SDS-PAGE safely in a high school laboratory was initially explored.

[![](https://static.igem.wiki/teams/5595/notebook/bba-25yslutm-map.avif)](https%7C//registry.igem.org/parts/bba-25yslutm)

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## What Should this Page Contain?

---

Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

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## Experimental Outcomes

#### Plasmid Construction and Preliminary Verification Process

[![](https://static.igem.wiki/teams/5595/notebook/bba-25yslutm-map.avif)](https%7C//registry.igem.org/parts/bba-25yslutm)

In 2025, we attempted to construct two plasmids: one was a dsRNA plasmid targeting superfold green fluorescence protein (sGFP), and the other was an expression plasmid for chitinase driven by the T7 promoter ([BBa\_25YSLUTM](https://registry.igem.org/parts/bba-25yslutm)).

For safety reasons, we cannot culture the pathogenic fungi that cause peach gummosis, and thus cannot verify whether the designed dsRNAs can interfere with their growth.

Instead, if we can develop dsRNA that interferes with the synthesis of sGFP, we will be able to determine the success of the experiment with the naked eye.

So a dsRNA plasmid was constructed to silence sGFP, providing us with a visible indicator of RNA interference efficiency to confirm the experimental workflow used for dsRNA design and plasmid construction. Meanwhile, the T7-driven peach chitinase plasmid aimed to express an enzyme predicted to be capable of degrading fungal chitin. Both constructs were transformed into *E. coli* DH5α for amplification.

Subsequently, plasmid DNA was extracted and verified by restriction enzyme digestion and Sanger sequencing. The results showed that only part of the dsRNA was inserted into the vector, but Sanger sequencing confirmed the correct insertion of the full-length peach chitinase. Therefore, pET-CH1 was transformed into *E. coli* BL21 for amplification and induced for expression.

#### Protein Expression Analysis and Optimization Directions

Total proteins from different IPTG treatments were harvested and analyzed by SDS-PAGE. Faint bands indicated partial expression, and further optimization of the protein extraction protocol, induction time, and IPTG concentration is needed to improve yield.

Although we did not obtain the expected clear bands for robust expression, the established experimental workflow—from vector construction to transformation and expression—provides a foundational framework for future attempts. The partial success in constructing the pET-CH1 plasmid demonstrates the feasibility of our cloning strategy, and we successfully performed SDS-PAGE analysis under limited conditions, confirming that our system can support protein expression with further refinement.

Currently, the laboratory is in continuous development, with ongoing efforts to enhance technical capabilities and infrastructure. We are actively optimizing expression conditions, including temperature, induction duration, and lysis buffers, to improve the solubility and detection of recombinant proteins. We may collaborate with other institutions or universities to conduct nickel-affinity chromatography purification trials and further validation via Western blotting, which are essential steps to verify the identity and functionality of the expressed protein.

## Discussion

With the cost reduction of gene sequencing, thousands of species' genomes have been decoded, but gene function annotation remains a critical gap. Our project addresses this by:

* Exploring unannotated gene value (e.g., peach chitinase)
* Developing a simple, high school-friendly protocol to popularize gene function verification.

#### **Plans**

* **AI-based Functional Prediction and Experimental Validation of the PpCH1**

  Use AI tools (e.g., AlphaFold for structure prediction, BLAST for homology analysis) to hypothesize PpCH1’s role in fungal cell wall degradation. Validate via enzymatic activity assays (e.g., chitin hydrolysis experiments).
* **Chitinase Extraction Workflow**

  Standardize steps for microbial/plant chitinase: sample pretreatment-centrifugation-column purification-activity detection. Compare processes by purity (SDS-PAGE) and yield (BCA assay) to enable industrial application.
* **Optimization of the dsRNA Fragment Design and Validation Workflow**

  dsRNA plays a core role in RNA interference technology. We will combine the latest bioinformatics algorithms and experimental data to optimize dsRNA design parameters, such as sequence length, GC content, and target region selection, in order to improve the silencing efficiency of dsRNA on target genes and reduce off-target effects. At the same time, we will establish a fast and accurate dsRNA validation system.

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