Notebook - Project Timeline

Our Project Notebook

"A journey of discovery, documented step by step."

Project Overview

Our team members conducted in-depth research on disease backgrounds, treatment methods, and drug selection, and determined our technical route after reviewing a large amount of literature. The usage method of our engineered bacteria is as follows: First, take an enteric-coated capsule made of calcium alginate shell orally. It will gradually disintegrate in the intestine and release the engineered bacteria. These engineered bacteria will grow on the intestinal mucosa and activate the delivery module.

This module senses DNA damage in the high oxidative stress microenvironment of colorectal cancer through the SOS promoter, activates the expression of the LuxI gene, and generates AHL signal molecule synthase. When AHL accumulates to a certain threshold, it binds to LuxR, triggers the Lux promoter, drives the expression of SRRz lytic protein, and leads to bacterial lysis. At this time, the IL-24 drug continuously expressed by the J23100 promoter is released. At the same time, EGFP produces green fluorescence, which facilitates our monitoring of drug release status in in vitro experiments.

Next, TRACER comes into play. The TRACER protein contains BRII, a sensitive peptide, and an anionic shielding peptide. Under the action of MMP-9 protease in the tumor microenvironment, the sensitive peptide is cleaved, and the anionic shielding peptide falls off, releasing BRII. BRII, with its positive charge, can bind to negatively charged gangliosides on the surface of cancer cells, accurately delivering IL-24 into the interior of cancer cells. IL-24 triggers cancer cell apoptosis by regulating the synergistic action of multiple signaling pathways, achieving efficient and low-toxic anticancer effects.



April - June: Initial Research Phase

April - June 2024

Research and Planning

During this period, our team focused on comprehensive research and strategic planning. We explored disease backgrounds, evaluated various treatment approaches, and carefully selected our drug candidates based on extensive literature review.

  • Disease Background Research: Conducted thorough analysis of colorectal cancer pathology, current treatment limitations, and potential therapeutic targets.
  • Treatment Methods Analysis: Evaluated conventional chemotherapy, targeted therapy, and immunotherapy approaches to identify gaps in current treatment paradigms.
  • Drug Selection: Reviewed IL-24 properties and selected it as our therapeutic payload based on its multi-pathway anti-cancer mechanisms.
  • Technical Route Design: Developed our dual-module system combining tumor-sensing drug release with activatable cell-penetrating peptide delivery.


July - October: Experimental Verification

The experimental verification of constructing strains can be roughly divided into three stages:

Objective: Construction of BL21(DE3)-pET-28a(+)-TRACER-IL24 strain and BL21(DE3)-pUC19-TRACER-IL24 strain

Key Procedures:

  • Plasmid mini-extraction preparation: Extracting pET-28a(+) plasmid and pUC19 plasmid
  • PCR amplification: Amplification of TRACER gene fragment
  • Enzyme digestion: Restriction enzyme digestion of plasmids and PCR products
  • Agarose gel electrophoresis: To detect target fragments for subsequent recovery
  • Gel extraction: Recovery of target fragments from agarose gel
  • DNA ligase-mediated fragment ligation: Linking TRACER to plasmid
  • Transformation: Transform the constructed plasmid into the BL21(DE3) strain

Objective: Extraction, purification, and electrophoresis verification of fusion protein

Key Procedures:

  • Large-scale cultivation: The BL21(DE3)-pET-28a(+)-TRACER-IL24 strain obtained through large-scale cultivation
  • Ultrasonic disruption and protein extraction: Cell lysis and initial protein extraction
  • Protein purification: Ni-NTA resin column purification
  • Protein concentration determination: To verify whether the target protein concentration is sufficient for subsequent experiments
  • SDS-PAGE electrophoresis: To verify whether there is target protein in the purified product

Objective: Experimental verification of the properties of fusion protein

Key Procedures:

  • MTT assay: The MTT assay was used to detect the changes in cell viability after co-culturing cells with different concentrations of TRACER-IL24 protein
  • Cancer cell killing efficacy: Evaluation of the therapeutic potential of the fusion protein


Detailed Experimental Timeline

July 11-17
Initial Construction Attempts

We attempted to construct the pET-28a(+)-TRACER-IL24 plasmid and validate the experimental steps and explore the reaction conditions through preliminary experiments.

Outcomes: Successfully established baseline protocols and identified optimal reaction conditions for subsequent experiments.

July 17-19
Target Plasmid Construction

Construct the target plasmid. Successfully obtained DNA fragments that can be used for subsequent experiments, and after double enzyme digestion and enzyme ligation, the pET-28a(+)-TRACER-IL24 plasmid and pUC19-TRACER-IL24 were obtained.

Key Achievement: Successful construction of both expression vectors containing the TRACER-IL24 fusion construct.

July 19-25
Transformation and Initial Protein Expression

Transform the obtained pUC19-TRACER-IL24 plasmid and pET-28a(+)-TRACER-IL24 plasmid into the competent BL21(DE3) strain, plate and culture, pick up the well-grown strain and inoculate it into LB medium and store it in glycerol.

Ultrasonically break the bacterial cells, extract total protein, and perform SDS-PAGE electrophoresis on the extract. Store the supernatant and sediment after crushing and centrifugation at -20℃.

Results: Successfully established stable recombinant strains and initiated protein expression studies.

July 26-28
Protein Induction Optimization

We conducted experiments involving protein induction expression, cell lysis extraction, and verification. Through preliminary experiments, we validated the experimental procedures and explored the reaction conditions.

Focus: Optimization of IPTG induction conditions and cell lysis parameters.

August 8-12
First Purification Attempt

Cultivate the BL21(DE3)-pET28a(+)-TRACER-IL24 strain, extract and purify the protein, and measure its concentration. The protein concentration was too low to yield visible bands in SDS-PAGE.

Challenge: Low protein yield necessitated optimization of expression and purification protocols.

August 12-14
Concentration Enhancement Attempts

Increasing the induction concentration and using freeze-drying, heating evaporation, and salting-out methods to concentrate the purified product, the concentration was measured. The protein concentration was still not sufficient for SDS-PAGE electrophoresis.

Approach: Tested multiple concentration methods including physical and chemical approaches.

August 14-19
Scale-Up and Dual Purification

The BL21(DE3)-pET-28a(+)-TRACER-IL24 strain was expanded and cultured, and the protein was extracted. The supernatant and precipitate were purified. The protein concentration was still insufficient for SDS-PAGE electrophoresis. The purified product was concentrated again, but the result was still unsatisfactory.

Strategy: Attempted purification of both soluble and insoluble fractions to maximize yield.

August 19-25
Buffer Optimization

We adjusted the amount of purification elution buffer and lysis buffer and repeated the experiment. The results were still not satisfactory. Although the protein concentration of the purified product after concentration was higher than previous times, it was still not sufficient for SDS-PAGE electrophoresis.

Learning: Identified the need for alternative expression systems or protein synthesis approaches.



Journey Reflections

Our experimental journey from April to October has been filled with challenges, learning experiences, and ultimately, significant achievements. Despite facing numerous obstacles during protein purification, our team demonstrated resilience and adaptability by exploring alternative approaches and maintaining focus on our ultimate goal.

Moving forward, we aim to further test and optimize our engineered bacterial delivery system(TRACER ＆ Deliver) and conduct more comprehensive studies to advance this promising therapeutic platform toward clinical translation.