Overview

To record the daily wetlab progress of our team and analyze the experimental results timely, we log the experimental contents for every experiment, and finally organize them. The compiled notebook is as follows.

Week 3, November 2024

The Initial Planning for 2025

• Promote our team across UCAS and begin recruitment efforts.
• Deliver introductory presentations to interested students.

Week 4, November 2024

The Initial Planning for 2025

• Conduct recruitment interviews
• Finalize the selection of new members.

Week 1, December 2024

The Initial Planning for 2025

• Organize Ice-breaking activities to integrate the new members into the team.

Week 3, December 2024-Week 2, January 2025

Training for New Members

• Presented reviews of wikis from previous iGEM teams to familiarize new members with the competition.
• Provide theoretical and experimental wetlab training for new members.

Week 1, March-Week 3, April 2025

Brainstorming

• Gather project ideas from winter brainstorming.
• Introduce the concept of a proteolysis-activated logic gate as a potential component of our project.

Week 4, April-Week 2, May 2025

Further Brainstorming and Concept Development

• Selected tomato and Pseudomonas syringaetomato pv. tomato (Pst) as the central elements of the project.

Week 3, May-Week 2, June 2025

Pathway Design

• Define the overall framework and route for our project.
• Establish the experimental verification plan for the project.

Week 3, June 2025

Pathway Design, Transformation, and Fluorescence Detection

• Conduct further background research on trehalase.
• Complete the initial design for most plasmids.
• Design primers for DNA cloning.
• Transform the constructed plasmids into E coli. BL21(DE3) strain competent cells.
• Carry out preliminary gene expression tests.

Week 4, June 2025

Pathway Design and Transformation

• Perform additional background research on AvrRpt2.
• Transform constructed plasmids into BL21(DE3).
• Clone DNA fragments via PCR.

Week 2, July 2025

Pathway Design and Plasmid Construction

• Conduct further background research on the second pathway.
• Transform constructed plasmids into the E coli. Top 10 and BL21(DE3) strains.
• Apply the overlap PCR technique to assemble DNA fragments.
• Perform homologous recombination for plasmid construction.

Week 3, July 2025

Project Finalization, Plasmid Construction and Validation

• Finalize the design of the second pathway.
• Transform DNA fragments into Top 10 and BL21(DE3).
• Utilize overlap PCR and touchdown PCR techniques to construct plasmids.
• Send constructed plasmids for sequencing.

Week 4, July 2025

Plasmid Redesign, Construction, and Protein Preliminary Expression

• Redesign the core plasmids of split_GFP.
• Verify the transformation via PCR.
• Construct plasmids applying the overlap PCR technique.
• Induce gene expression with IPTG.

Week 1, August 2025

Induction and Protein Purification

• Continue the induction.
• Verify expression by SDS-PAGE.
• Purify protein with Ni-NTA affinity chromatography.
• Construct plasmids via overlap PCR and homologous recombination.

Week 2, August 2025

Bioluminescence Detection, Validation.

• Detect the bioluminescence signal from split_Luc.
• Verify the protein expression by performing SDS-PAGE.

Week 3, August 2025

Induction and Protein Purification

• Continue the induction of split_GFP.
• Purify nTEVp utilizing Ni-NTA affinity chromatography.

Week 4, August 2025

Plasmid Redesign, Construction, Induction, and Protein Purification.

• Redesign the insoluble proteins with an MBP-tag added.
• Construct the trehalase plasmid (TreA) by homologous recombination.
• Re-induce split_GFP expression.
• Continue ongoing protein purification.

Week 1, September 2025

Validation

• Verify the transformation by colony PCR.

Week 4, September 2025

Plasmid Redesign, Reconstruction, and Validation

• Redesign the plasmids with AvrRpt2 cleavage sites.
• Verify TreA transformation by PCR.
• Reconstruct the plasmid with cTreA.

Week 1, October 2025

Wiki Freeze, Presentation Video, and Protein Purification

• Utilize varied methods for the purification of proteins in the pellet.

Full Records

For more detailed notebook description, please download the PDF of our lab record on Benchling.