iGEM 2025 — Team bit

![](https://static.igem.wiki/teams/5623/images/intro/wet-lab.avif)

# Our Project Notebook

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

* [Overview](#overview)
* [April - June](#april-june)
* [July - October](#july-october)
* [Timeline Details](#timeline-details)
* [Conclusion](#conclusion)

## Project Overview

![](https://static.igem.wiki/teams/5623/static/images/description/figure4.webp)

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:

## Stage 1Construction of Recombinant Strains

**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

## Stage 2Protein Extraction and Verification

**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

## Stage 3Functional Property Verification

**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: Construction and Initial Expression

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: Purification Challenges

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.

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![Engineering banner](https://static.igem.wiki/teams/5623/static/images/intro/engineering.avif)

# Engineering

"Building Biology, Cycle by Cycle."

* [Cycle 1](#cycle1)
* [Cycle 2](#cycle2)
* [Cycle 3](#cycle3)
* [Cycle 4](#cycle4)
* [Cycle 5](#cycle5)

## Cycle 1:

## Design and Acquisition of Fusion Proteins

## Design

The TRACER protein is composed of BRⅡ, a MMP9-Specific Cleavable Sensitive Peptide, and a Negative Charge Sequence.The design of this section is detailed in [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D).

##### Selection of Sensitive Peptides

To target colorectal cancer, our modeling systematically analyzed the transcriptome data of colorectal cancer and normal tissues, and identified significantly upregulated matrix metalloproteinase (MMP) family and their related pathways, as well as the sensitive peptide sequence PLGLAG that can be cleaved in the middle by MMP-2/9 enzymes.

We selected GSE10950 as the training set and downloaded the supplementary data of the independent external dataset "Transcriptomic and Cellular Content Analysis of Colorectal Cancer by Combining Multiple Independent Cohorts" as the validation set, and used UltraEdit and Excel for data processing. With the help of the Sangerbox platform, we performed differential expression gene screening, gene set enrichment analysis, PPI network and validation set analysis, and obtained the following important guiding conclusions:

**1. There are significant differences in gene expression patterns between colorectal cancer tissues and normal tissues.**

![Differential Expression Heatmap](https://static.igem.wiki/teams/5623/static/images/engineering/figure1.webp)

Fig.1 Differential Expression Heatmap

**2. The expression of MMP-9 enzyme in colorectal cancer tissues is significantly upregulated compared with normal tissues, while the difference in MMP-2 enzyme does not reach statistical significance.**

![Gene Enrichment Analysis](https://static.igem.wiki/teams/5623/static/images/engineering/figure2.webp)

Fig.2 Boxplot of Gene Enrichment Analysis for MMP9 vs. MMP2 (MMP9 on the left and MMP2 on the right)

**3. MMP-9 enzyme can serve as a molecular marker for colorectal cancer.**

Therefore, we chose to use PLGLAG as the CRC-targeting sensitive peptide, which constitutes a part of TRACER.

##### IL-24 as Therapeutic Molecule

To test the ability of Tracer to carry real anti-cancer drugs, we selected IL-24 as the therapeutic molecule. After entering cancer cells, IL-24 can trigger endoplasmic reticulum stress and initiate multi-pathway synergistic killing:

1. Inducing the expression of GADD family genes, phosphorylating eIF2α, activating the caspase cascade, and inducing cell apoptosis.
2. Upregulating Bax/Bak, inhibiting Bcl-2/Bcl-xL, releasing cytochrome c, and inducing mitochondria-dependent apoptosis.
3. Blocking PI3K/AKT, STAT3, and Src signals, inhibiting VEGF/TGF-β, and suppressing tumor angiogenesis, cancer cell proliferation and metastasis.
4. Stimulating T/NK cells to secrete IFN-γ and TNF-α, and enhancing immune recognition.

##### Molecular Docking and Dynamics Simulation

The molecular weight of the entire fusion protein TRACER-IL24 is 23.8 kDa. Considering that the relatively large molecular weight may affect the cleavage process of the sensitive peptide, we selected the human MMP9 crystal structure (PDB: 5I12) and the fusion protein TRACER-IL24 (Alphafold Predicted) to simulate and analyze the MMP-9 enzyme cleavage process through molecular docking and molecular dynamics. GROMACS was used to evaluate whether the catalytic geometry is accessible for a long time and whether the interface interaction is stable.

![Docking Conformation](https://static.igem.wiki/teams/5623/static/images/engineering/figure3.webp)

Fig.3 Docking Conformation of MMP9 and TRACER-IL24

![Molecular Dynamics Results](https://static.igem.wiki/teams/5623/static/images/engineering/figure4.webp)

Fig.4 Molecular Dynamics Simulation Results

**Key Findings:**

1. The number of hydrogen bonds and atomic contacts at the binding interface between MMP-9 enzyme and the sensitive peptide increases with simulation time and tends to stabilize (a-b).
2. The deeply buried interface of the enzymatic cleavage complex continues to increase and stabilizes, indicating that the enzymatic cleavage active interface has been formed (c).
3. The RMSD rises rapidly and then converges to a plateau, suggesting that the system has completed structural adjustment and remains stable (d).

Structural prediction of the entire TRACER fused with IL24 shows that MMP-9 enzyme can still recognize and stably bind to the sensitive peptide segment PLGLAG, providing solid theoretical support for our TRACER design.

##### Design of Negative Charged Shielding Peptide

To enable the designed negative charge sequence to achieve the shielding function, we collaborated with the modeling team to conduct predictions from two aspects: structure and shielding ability.

###### Structural Design

Three candidate schemes were proposed:

| Type | Sequence | Figure Reference |
| --- | --- | --- |
| Repeated Negative Charge Sequence | E×n | Fig.5(a) |
| Helical Hydrophobic | EXXEEXXE | Fig.5(b) |
| Flexible | EEG | Fig.5(c) |

After analysis, we chose to add glycine (Gly) to the repeated sequence to enhance flexibility. As shown in Fig. 5(c), the negative charge sequence (Glu-3, Glu-5, Glu-8) was found to effectively bind to the positively charged residues of BRⅡ (ARG-25, ARG-28, ARG-32).

![Negative Charge Structure Design](https://static.igem.wiki/teams/5623/static/images/engineering/figure5.webp)

Fig.5 Visualization of Negative Charge Structure Design

###### Prediction of Shielding Ability

We proposed a transmembrane ability evaluation paradigm termed **TRF (Transmembrane Readiness Framework)**:

![TRF Framework](https://static.igem.wiki/teams/5623/static/images/engineering/figure6.webp)

Fig.6 TRF Hierarchical Indicator Diagram

The evaluation integrates 8 indicators across three levels: Geometry & Interaction, Thermodynamics, and Kinetics. Results showed that the cleaved state (Cut) exhibited more compact structure, closer proximity to membrane, deeper insertion, favorable orientation, smaller energy barrier, and faster rearrangement compared to the uncleaved state (Total).

| TRF Level | Indicator | Total（Uncleaved） | Cut（Cleaved） | Conclusion |
| --- | --- | --- | --- | --- |
| Geometry & Interaction | RMSD | Stable (0.02–0.025 nm) | Stable | Both are stable. |
|  | Rg | 1.94 nm | 1.77 nm | Cut is more compact (facilitates membrane approach). |
|  | Minimum Distance Between Centroid and Membrane | ~2.0–2.2 nm | ~1.6 nm | Cut is closer to the head group layer. |
|  | Insertion Depth (Smaller value = Deeper insertion) | ~6.3 nm | ~5.7 nm | Cut inserts deeper. |
|  | Tilt Angle Distribution/Temporal Profile | ~99°（horizontal lying） | ~155°（oblique insertion/near-vertical） | Cut orientation is favorable for transmembrane penetration. |
| Thermodynamics | SMD Force-Displacement Slope/Plateau | Steep slope, no plateau | Low slope, obvious segmented plateau | Cut has a smaller effective energy barrier. |
|  | SMD Force-Time/Displacement-Time | Early saturation, limited displacement | Continuous progression, linear increase in displacement | Cut is more likely to overcome the energy barrier. |
| Kinetics | Orientation-Related Time Constant (τ) | ~0.49 ns | ~0.12 ns | Cut shows faster rearrangement and stronger adaptability. |

##### Initial Genetic Circuit Design

Initially, we planned to use Escherichia coli (E. coli) as the pharmaceutical production host for the production of the fusion protein, and combine the fusion protein with our self-designed microneedle system for drug delivery.

![Genetic Circuit](https://static.igem.wiki/teams/5623/static/images/engineering/figure7.webp)

Fig.7 Genetic Circuit Diagram of TRACER-IL24

In our design, the TRACER protein consists of a negatively charged sequence (red), a cleavage-sensitive peptide (green), and the cell-penetrating peptide BRⅡ (blue). Under normal conditions, the negatively charged sequence reduces the positive charge on the protein surface, rendering the protein unable to enter cells via macropinocytosis. In the colorectal cancer (CRC) tumor microenvironment (TME), the highly expressed matrix metalloproteinase MMP-9 will cleave the cleavage-sensitive peptide, leading to the detachment of the negatively charged sequence. Subsequently, the cell-penetrating peptide delivers the therapeutic agent IL-24 into cancer cells to exert its therapeutic effect.

## Construction

**Genetic Circuit:** All fusion protein genes were entrusted to Thermo Fisher for synthesis in accordance with the Biobrick standard. Subsequently, the gene fragments were amplified via PCR, followed by restriction enzyme digestion of the PCR-amplified products. Thereafter, the obtained fragments were analyzed using nucleic acid gel electrophoresis, and the electrophoretic products were purified and recovered by the gel extraction method. Finally, the purified genetic circuit fragments and the empty pET-28a(+) vector were subjected to DNA ligation in a 37°C incubator, with incubation for 24 hours.

![Nucleic Acid Electrophoresis pET-28a](https://static.igem.wiki/teams/5623/static/images/engineering/figure8.webp)

Fig.8 Verification Results of Nucleic Acid Electrophoresis (pET-28a(+))

![Nucleic Acid Electrophoresis pUC19](https://static.igem.wiki/teams/5623/static/images/engineering/figure9.webp)

Fig.9 Verification Results of Nucleic Acid Electrophoresis (pUC19)

After performing Polymerase Chain Reaction (PCR) on the target gene fragment, a restriction endonuclease digestion assay was conducted between the PCR product and the full-circuit cloning vector, and the digested fragments were analyzed based on agarose gel electrophoresis.

**Fig.8 Results:** Lane 1 is the standard nucleic acid molecular Marker Ⅲ; Lane 2 is the pET-28a(+) plasmid vector with a theoretical length of 5369 bp; Lanes 3-4 are the pET-28a(+) plasmid fragments after double digestion with a theoretical length of 5350 bp; Lanes 5-6 are the target fragments obtained after PCR with a theoretical length of 697 bp, and all lengths are consistent with the expected values.

**Fig.9 Results:** Lane 1 is Marker Ⅲ; Lanes 2-3 are the pUC19 plasmid fragments after double digestion with a theoretical length of 2635 bp; Lanes 4-5 are the target fragments obtained via PCR with a theoretical length of 697 bp; Lane 6 is the pUC19 plasmid vector with a theoretical length of 2686 bp, and all lengths are consistent with the expected values.

The successfully constructed plasmids were transformed into BL21(DE3) competent cells, which were then cultured on LB solid medium containing kanamycin sulfate, and finally single colonies harboring the recombinant plasmids were obtained.

![Transformation Results](https://static.igem.wiki/teams/5623/static/images/engineering/figure10.webp)

Fig.10 Screening Results of Recombinant Plasmid Transformation into BL21(DE3)

## Test

The bacterial culture and kanamycin were added to the LB medium at a ratio of 1:100 and 1:1000 respectively, and incubated at 37°C until reaching the logarithmic growth phase with an OD600 value of 0.4-0.6. After that, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM for overnight induction. Subsequently, the bacterial cells were sonicated for lysis, followed by centrifugation to collect the supernatant. The protein was purified from the supernatant using affinity chromatography, and the protein concentration was determined by the BCA method. Finally, the molecular weight of the protein was characterized via SDS-PAGE.

![BCA Assay Results](https://static.igem.wiki/teams/5623/static/images/engineering/figure11.webp)

Fig.11 Results of Protein Concentration Determination by BCA Assay

![SDS-PAGE Results](https://static.igem.wiki/teams/5623/static/images/engineering/figure12.webp)

Fig.12 Results of Protein Molecular Weight Characterization by SDS-PAGE

The standard curve determined and plotted by the BCA assay is shown in Fig.11. Substituting the measured data, the maximum concentration of the purified samples is approximately 179.4 μg/ml. In SDS-PAGE, no distinct bands were observed except for the Marker shown in Fig.12.

The results of the BCA assay and SDS-PAGE indicated that the extracted protein had a low concentration, failing to show distinct bands in SDS-PAGE and thus unable to meet the requirements of subsequent experiments.

## Learn

In multiple rounds of construction verification and protein expression experiments, recombinant plasmids and engineered bacteria were successfully obtained; however, proteins with sufficient concentration could not be purified. We speculate that this may be due to the excessively low expression level of the constructed recombinant plasmids. Therefore, we plan to increase the intensity of induced expression and perform concentration after protein extraction to raise the protein concentration.

## Cycle 2:

## Increased Induction Intensity and Concentration

## Design

Based on the results of the previous cycle, where recombinant plasmids and engineered bacteria were successfully constructed but the concentration of the extracted protein was insufficient, we decided, after investigation, to increase the concentration of IPTG induction and adopt three methods—water bath concentration, ammonium sulfate precipitation concentration, and freeze-drying concentration—to enhance the protein concentration.

## Construction

After obtaining the engineered bacteria, the bacterial culture and kanamycin were added to fresh LB medium at ratios of 1:100 and 1:1000, respectively, for logarithmic growth. Unlike the previous cycle, a final concentration of 1 mM IPTG was then added for overnight induction, followed by disruption of bacterial cells by ultrasonication. The supernatant was collected by centrifugation, and proteins were purified by affinity chromatography.

###### Three Concentration Methods:

1. **Water bath concentration:** The obtained purified samples were placed with lids opened and incubated in a 40°C water bath until approximately 1/10 of the liquid remained.
2. **Ammonium sulfate concentration (pellet):** A saturated ammonium sulfate solution was prepared under ice bath, and slowly added to the purified samples in two portions at a ratio of 3:2, with precipitation formed during the process. The mixture was inverted to mix while adding. After 30 minutes of ice bath, centrifugation was performed to remove the supernatant. The precipitate was resuspended again with saturated ammonium sulfate solution, followed by another centrifugation to remove the supernatant, and then resuspended with 1-3 mL TBS (20 mM Tris, 0.137 M NaCl, pH 7.6±0.1 (25°C)).
3. **Freeze-drying concentration:** The obtained purified samples were placed in a lyophilizer and lyophilized at -40°C and 500 Pa for 12 hours.

## Test

After concentration, the protein content in the purified sample was determined using the BCA method:

![BCA Assay Cycle 2](https://static.igem.wiki/teams/5623/static/images/engineering/figure13.webp)

Fig.13 Results of Protein Concentration Determination by BCA Assay

The standard curve determined and plotted by the BCA method is shown in Fig.13. By substituting the measured data, it was found that among the three concentration methods, freeze-drying concentration showed better concentration efficiency. However, the maximum concentration of the concentrated and purified sample was approximately 114.7 μg/ml, which still failed to meet our requirements.

The results of the BCA method indicated that after enhanced induction and the application of the three concentration methods, the protein concentration did not show a significant increase.

## Learn

After multiple rounds of repeated experiments, we found that no high-concentration protein was detected in the supernatant even after concentration. At this point, the results from the modeling group indicated that the fusion protein has low solubility and may be present in the precipitate. Therefore, we plan to separately extract the protein from the supernatant and the precipitate after sonication.

![Solubility Analysis](https://static.igem.wiki/teams/5623/static/images/engineering/figure14.webp)

Fig.14 Modeling Analysis of TRACER-IL24 Protein Solubility

## Cycle 3:

## Attempt to Extract Fusion Protein from the Pellet

## Design

In the previous cycle, the experimental results and data from the modeling group enlightened us that the fusion protein may exist in the precipitate after sonication due to its properties. Therefore, we plan to extract proteins from both the supernatant and the precipitate to obtain the fusion protein.

## Construction

After sonication, we extracted proteins from the supernatant. Subsequently, the pellet was resuspended in lysis buffer and subjected to sonication again. After centrifugation, the pellet was collected, resuspended in buffer, and then mixed with protein denaturation mixture followed by another round of sonication. After centrifugation, the supernatant was collected. This process was repeated twice to obtain the purified product.

## Test

The protein content in the purified sample was determined using the BCA method:

![BCA First Experiment](https://static.igem.wiki/teams/5623/static/images/engineering/figure15-1.webp)

First experimental result

![BCA Second Experiment](https://static.igem.wiki/teams/5623/static/images/engineering/figure15-2.webp)

Second experimental result

Fig.15 Results of Protein Concentration Determination by BCA Assay

The standard curves determined and plotted by the BCA assay are shown in Figs.15. Substituting the measured data, it was found that the maximum concentration of the purified samples increased, with the highest concentration observed in the supernatant (approximately 247.6 μg/ml), which still failed to meet the requirements of subsequent experiments. Results of the BCA assay indicated that neither the supernatant nor the precipitate exhibited a high protein concentration, thus failing to satisfy the demands of subsequent experiments.

## Learn

Experiments and learnings from the previous cycles have shown that although plasmids were successfully constructed, transformed, and engineered strains meeting the requirements were screened, no high-concentration protein was extracted from either the supernatant or pellet of ultrasonic disruption. This suggests that there may be issues in gene expression or protein extraction steps.

**Regarding the above phenomenon, combined with the work of the modeling team and the results of our literature research, we propose the following possibilities:**

1. The solubility of the fusion protein is low, leading to excessive production of inclusion bodies during expression. The protein expression process needs optimization.
2. There is a lack of library construction and experimental screening for combinations between promoter strength, RBS, and the expressed gene.
3. The molecular weight of the selected IL-24 protein may be excessively large.

**In the future, we plan to make the following modifications in the biological route:**

1. Add fusion-promoting tags to improve solubility and facilitate protein production;
2. Construct a screening library of promoter-RBS-expressed gene for high-throughput screening.

*However, due to time constraints, we did not conduct subsequent experiments and optimizations.*

## Cycle 4:

## Test the Cytotoxicity of Fusion Protein Against Cancer Cells

## Design

Since we failed to obtain the fusion protein with sufficient concentration in the previous cycles, we entrusted Beijing Xinxinsantatech Co. Ltd. to help us synthesize the fusion protein. We treated cancer cells with the obtained protein and detected the cytotoxicity of the fusion protein to cancer cells by evaluating cell viability.

## Construction

Due to sterilization issues in the incubator, we were unable to conduct the experiment before the wiki deadline. It will be carried out before the Jamboree Presentation.

## Cycle 5:

## Redesign the Genetic Circuit

Escherichia coli serving as both factories and drug delivery carriers

## Design

After conducting the aforementioned experiments, the protein expression process we currently have is insufficient to produce a sufficient quantity of protein for microneedle drug loading. Therefore, we plan to engineer *Escherichia coli* to serve as a carrier for oral drug delivery, enabling it to both fulfill the production function and release the drug. We referenced the four-module design of the oscillatory lysis circuit described in the study "*Synchronized cycles of bacterial lysis for in vivo delivery*". Our aim is to avoid attempting colonization of the engineered bacteria; instead, we will achieve effective drug concentrations through regular administration. The engineered bacteria will reach the intestinal mucosa via enteric-coated capsules, grow there, and be shed along with the natural renewal of the mucosa. This design, combined with the microneedles in the hardware component, forms an **"In and Out"** delivery toolbox, which can significantly improve patient compliance.

To align with the therapeutic target of colorectal cancer, our research revealed that the intestinal tract of colorectal cancer patients exhibits extensive infiltration of inflammatory cells, leading to a significant increase in oxidative damage levels. Consequently, we selected an oxidative damage-activated promoter as the starting point for our activation module and employed the SRRz gene—which enables faster lysis—to trigger bacterial lysis.

![Genetic Circuit Deliver Module](https://static.igem.wiki/teams/5623/static/images/engineering/figure18.webp)

Fig.18 Genetic circuit of the deliver module

In this design, E. coli will directly enter the human body for therapeutic purposes. Pj23100 can constitutively express the fusion protein, while upon entering colorectal cancer tissues, E. coli will be induced to lyse, releasing the fusion protein for treatment.

![Deliver Circuit Schematic](https://static.igem.wiki/teams/5623/static/images/engineering/figure19.webp)

Fig.19 Deliver Circuit Schematic Diagram

## Construction

**Note:** Due to difficulties in plasmid synthesis and time constraints, we failed to complete subsequent experiments before the Wiki deadline. In the presentation at Jamboree, we will showcase our subsequent experimental results.

**Important:** It should be noted that the use of BL21(DE3) in experiments was solely for characterizing the function of the module. In future plans, the probiotic EcN will serve as the carrier for in vivo experiments.

## Test

Due to time constraints, we failed to complete the subsequent experiments before the Wiki deadline. We will present our subsequent experimental results at the Jamboree presentation. However, based on the Agent-Based Modeling (ABM) framework, we used NetLogo to simulate the lysis conditions under different inoculation doses.

![Effects of Inoculum Size](https://static.igem.wiki/teams/5623/static/images/engineering/figure20.avif)

Fig.20 Effects of different initial inoculum sizes on biomass accumulation and lysis cycle

![NetLogo Spatiotemporal Visualization](https://static.igem.wiki/teams/5623/static/images/engineering/figure21.webp)

Fig.21 Spatiotemporal visualization results of different inoculum sizes simulated by NetLogo
a. Num=2000; b. Num=5000; c. Num=8000; d. Num=10000
The top left shows curves of total biomass and products; the top right shows the spatial distribution map (red represents regions with high concentration of lysis proteins, and green represents regions of viable cells).

NetLogo simulations revealed the quantitative relationship between "inoculation dose--lysis cycle" at the tissue level: the initial inoculation dose exhibits a negative correlation with the lysis cycle. Within a certain range of inoculation doses, the higher the initial bacterial load, the more rapidly quorum sensing signals accumulate, and the shorter the lysis cycle; when the initial bacterial load is too low, the initiation of lysis is delayed, leading to a longer lysis cycle.

## Learn

In this cycle, we only redesigned and modified the gene circuit. However, combining the results from previous cycles and modeling, E. coli can effectively express the fusion protein and lyse and die after a certain period. Through constitutive expression combined with induced lysis for drug delivery, we believe it will be possible to produce sufficient drugs in the human body and achieve targeted therapy.

Meanwhile, the NetLogo simulation results provide a theoretical basis for applying engineered bacteria in practical therapeutic settings: by adjusting the initial dosage, precise regulation of the lysis rhythm can be achieved, thereby controlling the lysis cycle and obtaining the optimal therapeutic effect.

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Experimental Workflow

![Experimental Workflow banner](https://static.igem.wiki/teams/5623/static/images/intro/wet-lab.avif)

# Experimental Workflow

Detailed protocols and methodological design for TRACER system construction, expression, and purification.

* [Overview Flow](#overview)
* [Gene Acquisition](#1)
* [Plasmid Construction](#2)
* [PCR and Restriction Digestion](#3)
* [Gel Electrophoresis](#4)
* [DNA Fragment Ligation](#5)
* [Strain Construction](#6)
* [Protein Expression](#7)
* [Protein Purification](#8)
* [Protein Concentration](#9)
* [Concentration Measurement](#10)
* [SDS-PAGE](#11)
* [MTT Assay](#12)

## Overview Flow

[Gene Acquisition](#1)
[Plasmid Construction](#2)
[PCR and DNA Fragment Restriction Digestion](#3)
[Agarose Gel Electrophoresis and Gel Extraction](#4)
[DNA fragment enzymatic ligation](#5)
[Strain Construction](#6)
[Expression of protein](#7)
[Protein Purification](#8)
[Protein Concentration](#9)
[Protein Concentration Measurement](#10)
[SDS-PAGE](#11)
[Protein Characterization Experiments](#12)

## I. Gene Acquisition

TRACER-IL24 gene and Deliver gene were synthesized by Jiangsu Genecefe Biotechnology. in accordance with the Biobrick standard. Among these, the TRACER-IL24 gene was obtained as a linear fragment via PCR; the pET-28a(+) plasmid was amplified using the DH5α strain for high-copy replication and subsequently isolated via plasmid miniprep.

## II. Plasmid Construction

### Vector Acquisition

#### Materials

Constant Temperature Water Bath, Shaker Incubator, Centrifuge, Micropipette, Erlenmeyer Flask (150 mL), 1.5 mL Microcentrifuge Tube, Plasmid Miniprep Kit, Chemically Competent E. coli DH5α Cells, Solid LB Agar Medium supplemented with Kanamycin, Kanamycin Sulfate Solution (100 mg/mL), LB Medium.

#### Protocol

#### 1. Transformation

* Commercially available competent cells (100 μL) were thawed on ice. The ultra-clean bench UV lamp was turned on in advance and sterilized for 15 min. Subsequently, an alcohol lamp was lit within the ultra-clean bench. 50 μL of competent cells were pipetted into a new sterile centrifuge tube, and 5 μL of pET-28a(+) plasmid solution was added.
* 5 μL of plasmid was directly added to the remaining competent cells. The aforementioned centrifuge tubes containing the plasmid solution were placed on an ice box for 30 min. The water bath was pre-heated and set to 42°C. The ice-bathed centrifuge tubes were placed in the 42°C water bath for a heat shock of 45 s, immediately removed, and placed on the ice box for 2 min to complete the transformation process.
* Pre-aliquoted LB liquid medium without antibiotic was retrieved. 500 μL of the liquid medium was pipetted into the aforementioned heat-shocked centrifuge tube. The tube was incubated in a shaker at 37°C, 180 rpm for 1 h.
* The centrifuge tube was removed from the shaker and centrifuged at 5000 rpm for 30 s. Near the alcohol lamp, 400 μL of the supernatant was aspirated and discarded. The bacterial pellet was resuspended using a micropipette. The corresponding solid medium was selected based on the plasmid antibiotic resistance. Near the alcohol lamp, 100 μL of the resuspended bacterial culture was aspirated and added to the surface of the solid medium. The culture was spread evenly using a spreader. After covering the petri dish lid, the solid medium was inverted and placed in a 37°C constant temperature incubator for 12 h.
* A single colony with good growth was selected and inoculated into 30 mL of LB medium supplemented with 30 μL of Kanamycin sulfate solution, followed by overnight culture in a shaker at 37°C, 180 rpm.

#### 2. Plasmid Miniprep

* Add 500 μL of Equilibration Buffer BL to the CP3 adsorption column (place the column into the collection tube). Centrifuge at 12,000 rpm for 1 min. Discard the flow-through from the collection tube and place the CP3 column back into the collection tube.
* Take 1.5 ml of the overnight bacterial culture and add it to a centrifuge tube. Centrifuge at 12,000 rpm for 1 min, and carefully remove the supernatant.
* Add 250 μL of Solution P1 (already supplemented with RNase A) to the centrifuge tube containing the bacterial pellet. Resuspend the bacterial pellet completely using a pipette or a vortex mixer.
* Add 250 μL of Solution P2 to the centrifuge tube. Gently invert the tube 5-8 times to lyse the bacteria thoroughly.
* Add 350 μL of Solution P3 to the centrifuge tube. Immediately invert the tube gently 5-8 times to mix thoroughly. A white flocculent precipitate will form. Centrifuge at 12,000 rpm for 10 min.
* Carefully pipette the supernatant from the previous step and transfer it to the CP3 adsorption column (placed in a collection tube). Centrifuge at 12,000 rpm for 60 s. Discard the flow-through from the collection tube and place the CP3 column back into the collection tube.
* Add 600 μL of Wash Buffer PW (already supplemented with absolute ethanol) to the CP3 adsorption column. Centrifuge at 12,000 rpm for 60 s. Discard the flow-through from the collection tube and place the CP3 column back into the collection tube. Repeat this step once.
* Place the CP3 adsorption column into the collection tube and centrifuge at 12,000 rpm for 2 min to remove residual wash buffer.
* Open the lid of the CP3 adsorption column and let it stand at room temperature for several minutes to allow the residual ethanol to evaporate completely. Proceed with elution only after no alcohol odor remains.
* Place each CP3 adsorption column into clean centrifuge tubes. Carefully add 50 μL of Elution Buffer EB to the center of the adsorption membrane. Let it stand at room temperature for 2 min. Centrifuge at 12,000 rpm for 2 min to collect the plasmid solution in the centrifuge tube. Store the final product at -20°C.

## III. PCR and DNA Fragment Restriction Digestion

### Material

Thermocycler, 0.2 mL PCR tubes, Micropipettes, Incubator, 2× PCR mix, Forward primer, Reverse primer, Sterile water, DNA template, pET-28a(+) plasmid (miniprep product), Rcut Smart Buffer (10X), HindIII restriction enzyme, EcoRI restriction enzyme.

### Protocol

#### PCR

Two PCR tubes were prepared with the reaction components as specified (total volume 25 μL per tube).

| PCR system | Volume |
| --- | --- |
| 2× PCR Mix | 12.5 μL |
| Template DNA | 1.5 μL |
| Forward Primer | 1 μL |
| Reverse Primer | 1 μL |
| H₂O | 9 μL |
| Total | 25 μL |

* The tubes were placed in a thermocycler and subjected to the following program: initial denaturation at 94°C for 120 seconds; 30 cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 60 seconds; followed by a final extension at 72°C for 5 minutes.
* The PCR products were stored at -20°C.

#### Enzyme digestion

The restriction digestion was set up as a 20 μL reaction system.

| Digestion system | Volume |
| --- | --- |
| Rcut smart | 2 μL |
| DNA strain | 17 μL |
| Hind III | 0.5 μL |
| EcoR I | 0.5 μL |
| Total | 20 μL |

* All microcentrifuge tubes were subsequently incubated in a constant temperature incubator at 37°C for 2 hours.

## IV. Agarose Gel Electrophoresis and Gel Extraction

### Material

Constant temperature water bath, electronic balance, electrophoresis system, gel imaging analysis system, centrifuge, micropipettes, glass flask, microcentrifuge tubes (1.5 mL, 2 mL), agarose, electrophoresis buffer, fluorescent nucleic acid dye, UE DNA gel extraction kit, DNA loading buffer, nucleic acid standard marker.

### Protocol

#### Agarose Gel Electrophoresis

* 1.5% Agarose Gel Preparation: Measure 30 mL of 1x TAE electrophoresis buffer and add it to a glass bottle. Weigh 0.45 g of agarose and add it to the bottle to prepare a 1.5% agarose solution. Heat the mixture in a microwave oven until boiling. Allow the solution to cool at room temperature to approximately 60°C, then add 3 μL of fluorescent dye and mix thoroughly. Retrieve the gel casting tray and insert the comb. Pour the prepared gel solution into the tray and allow it to solidify at room temperature.
* Place the cooled and solidified agarose gel into the electrophoresis tank. Mix the restriction digestion product with DNA loading buffer at a 5:1 ratio, and load the mixture into the wells. Turn on the electrophoresis power supply and set the voltage to 110 V. Run the gel for 40 minutes.
* Remove the agarose gel. Analyze and document the electrophoresis results using a gel imaging analysis system.

#### DNA Fragment Gel Extraction

* Under UV illumination, excise the agarose gel slice containing the target DNA band. Blot the surface dry with laboratory tissue and mince the gel. Determine the gel weight (the weight of a 1.5 mL microcentrifuge tube should be tared in advance), treating this weight as the gel volume (where 100 mg = 100 μL).
* Add three gel volumes of Buffer DE-A to the minced gel and mix thoroughly. Incubate at 75°C, vortexing intermittently every 2-3 minutes until the gel is completely dissolved.
* Add 0.5 volume of Buffer DE-B relative to the volume of Buffer DE-A used, and mix thoroughly.
* Transfer the mixture to a DNA preparation column (assembled in a 2 mL collection tube). Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through. Reassemble the column with the collection tube, add 500 μL of Buffer W1, and centrifuge at 12,000 rpm for 30 seconds. Discard the flow-through.
* Place the column back into the collection tube, add 700 μL of Buffer W2, and centrifuge at 12,000 × g for 30 seconds. Discard the flow-through. Repeat the washing step with an additional 700 μL of Buffer W2, followed by centrifugation at 12,000 rpm for 1 minute.
* Reinsert the column into the collection tube and perform a final centrifugation at 12,000 rpm for 1 minute to remove residual wash buffer.
* Transfer the column to a clean 1.5 mL microcentrifuge tube. Apply 25-30 μL of Elution Buffer directly to the center of the column membrane. Allow it to stand at room temperature for 1 minute, then centrifuge at 12,000 rpm for 1 minute to elute the DNA.
* Store the final product at -20°C.

## V. DNA Fragment Enzymatic Ligation

### Material

PCR tubes, tube floats, foam rack, vector backbone, insert fragment, 10X DNA Ligase Buffer, nuclease-free water, T4 DNA Ligase.

### Protocol

| Ligation system | Volume |
| --- | --- |
| Vector fragment | 3 μL |
| Target fragment | 12 μL |
| DNA ligase buffer 10x | 2.5 μL |
| Nuclease-free Water | 6.5 μL |
| T4 DNA Ligase | 1 μL |
| Total | 25 μL |

Following the pipetting scheme outlined in the table above, the reaction mixtures in PCR tubes were placed in an ice-water bath for a 24-hour ligation reaction.

## VI. Strain Construction

### Transformation and Bacterial Survival

#### Material

Constant temperature water bath, shaker incubator, centrifuge, micropipettes, 150 mL Erlenmeyer flasks, 1.5 mL microcentrifuge tubes, pET-28a(+)-TRACER plasmid solution, E. coli BL21(DE3) competent cells, kanamycin-resistant solid LB agar medium, kanamycin sulfate solution (100 mg/mL), LB medium, 50% glycerol solution.

#### Protocol

* Thaw a 100 μL of commercially available competent cells on ice. Pre-sterilize the biological safety cabinet by UV irradiation for 15 minutes. Add plasmid solution to each tube at a 10:1 volume ratio (competent cells : plasmid solution).
* Place the microcentrifuge tubes containing the plasmid-cell mixtures on ice for 30 minutes. Preheat a water bath to 42°C. Following ice incubation, subject the tubes to a 42°C heat shock for 45 seconds, then immediately return them to ice for 2 minutes to complete the transformation.
* Retrieve pre-aliquoted antibiotic-free LB liquid medium. Add 500 μL of the medium to each heat-shocked microcentrifuge tube. Incubate the tubes in a shaker incubator at 37°C and 180 rpm for 1 hour for bacterial recovery.
* Centrifuge the tubes at 7,000 rpm for 30 seconds after incubation. Aseptically aspirate and discard approximately 400 μL of supernatant near an alcohol burner flame. Resuspend the cell pellet in the remaining medium. Retrieve solid LB agar medium supplemented with the appropriate antibiotic corresponding to the plasmid resistance marker. Aseptically spread 100 μL of the resuspended culture evenly onto the solid medium surface using a sterile spreader near the alcohol burner flame. Replace the Petri dish lid, invert the plate, and incubate at 37°C for 12 hours. Select well-isolated single colonies to inoculate 30 mL of LB liquid medium supplemented with 30 μL kanamycin sulfate solution, followed by overnight culture in a shaker incubator at 37°C and 180 rpm.
* Preserve bacterial strains at -20°C using the glycerol stock method by mixing 500 μL of bacterial culture with an equal volume of 50% glycerol solution per cryovial.

## VII. Expression of Protein

### Expand Cultivation and Protein Extraction

#### Material

Erlenmeyer flasks (150 mL), Shaker incubator, Centrifuge, Micropipettes, 1.5 mL microcentrifuge tubes, Ultrasonic cell disruptor, E. coli BL21(DE3) strain harboring the pET-28a(+)-Tracer plasmid, Double-distilled water (ddH₂O), Kanamycin sulfate solution (100 mg/mL), LB medium, IPTG solution (100 mg/mL), PBS buffer (pH 7.4), Protease inhibitor cocktail (50×), Ammonium bicarbonate, Urea.

#### Protocol

* Lysis Buffer Preparation: 50 mM Ammonium Bicarbonate, 8 M Urea, 1:50 Protease Inhibitor Cocktail.
* Retrieve the preserved bacterial strain and thaw it on ice. Add 30 μL of Kanamycin sulfate solution to each 30 mL culture medium bottle, and inoculate with 0.3 mL of the bacterial culture. Incubate in a shaker at 37°C, 180 rpm until the OD600 reaches between 0.4 and 0.6. Under a sterile workbench, add 30 μL of 100 mg/mL IPTG solution to each bottle. Subsequently, incubate the cultures in a shaker at 25°C, 180 rpm for 16 hours.
* Collect the bacterial culture and aliquot it into 15 mL centrifuge tubes. Centrifuge at 3500 rpm for 10 minutes.
* Discard the supernatant. Resuspend the cell pellet in 1 mL of PBS solution, then transfer the suspension to a 1.5 mL centrifuge tube. Centrifuge at 5000 rpm for 10 minutes.
* Repeat the previous step (PBS wash and centrifugation) once more. After the second wash, resuspend the final cell pellet in 1 mL of Lysis Buffer.
* Initiate the Ultrasonic Cell Disruptor. Disrupt the cell suspension using a probe sonicator on an ice-water bath with the following parameters: 2-second pulse ON, 2-second pulse OFF for 12 minutes until the bacterial suspension becomes clear and translucent. After disruption, centrifuge the lysate at 4°C, 12000 rpm for 20 minutes to separate the supernatant and pellet.
* Store the resulting products at -20°C.

## VIII. Protein Purification

### Material

Gravity flow column with upper and lower frits, Micropipettes, Centrifuge tubes, Retort stand, Ni-NTA resin, 10× TBS buffer, Imidazole, Urea, Triton X-100, Sample for purification.

### Protocol

#### Preparations

* Assemble the gravity flow column. Insert the bottom frit, and wash the column with 1 column volume (CV) of purified water. Add 2–4 mL of pre-mixed Ni-NTA resin slurry to the column. Open the bottom outlet and allow the storage solution to drain completely. Wash the resin with 5 CV of purified water, then insert the top frit. Equilibrate the prepared column with 5 CV of 1× TBS buffer.

#### Supernatant Protein Purification

The compositions of all solutions used for the purification process are detailed in the table below.

|  |  |  |  |
| --- | --- | --- | --- |
| Solutions | Tris | NaCl | Imidazole |
| Equilibration Buffer | 50 mM | 0.15 M | 20 mM |
| Elution Buffer Ⅰ | 50 mM | 0.15 M | 50 mM |
| Elution Buffer Ⅱ | 50 mM | 0.15 M | 100 mM |
| Elution Buffer Ⅲ | 50 mM | 0.15 M | 300 mM |

* All solutions mentioned above were prepared using 1× TBS buffer, pH 7.6.
* The sample was recirculated through the column more than 5 times. The column was then washed with 10-20 column volumes (CV) of Equilibration Buffer, and the final column volume of wash effluent was collected and retained for later analysis. Subsequently, the target protein was eluted using a gradient with 5 CV each of Elution Buffer I, II, and III, and the eluates were collected.

#### Precipitation Protein Purification

* Resuspend the pellet in Lysis Buffer (pH 8.0, 50 mM Tris, 2 mM EDTA, 150 mM NaCl, 20 mM imidazole, 1% Triton X-100). Disrupt the cell suspension using a probe sonicator on an ice-water bath with the following parameters: 3-second pulse ON, 8-second pulse OFF, for a total duration of 10 minutes.
* Centrifuge the lysate at 13,000 rpm for 20 minutes at 4°C. Collect the insoluble pellet.
* Solubilize the pellet obtained from centrifugation by first adding 1 mL of Wash Buffer (20 mM imidazole, 20 mM Tris, pH 8.0) per gram of pellet. Then, add 4-5 mL of Denaturation Buffer (50 mM Tris buffer, pH 8.0, containing 0.15 M NaCl and 8 M urea) per gram of pellet.
* Subject the mixture to probe sonication on an ice-water bath using the following cycle: 3-second pulse ON, 8-second pulse OFF until the suspension becomes clear and translucent.
* Centrifuge the sonicated lysate at 12,000 rpm for 10 minutes at 4°C. Collect the supernatant and subject it to a second centrifugation under the same conditions (12,000 rpm, 10 minutes, 4°C). Collect the final supernatant.

The compositions of all solutions used for the purification process are detailed in the table below.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Solutions | Tris | NaCl | Urea | Imidazole |
| Equilibration Buffer | 50 mM | 0.15 M | 8 M | 20 mM |
| Elution Buffer Ⅰ | 50 mM | 0.15 M | 8 M | 50 mM |
| Elution Buffer Ⅱ | 50 mM | 0.15 M | 8 M | 100 mM |
| Elution Buffer Ⅲ | 50 mM | 0.15 M | 8 M | 300 mM |
| Elution Buffer Ⅳ | 50 mM | 0.15 M | 8 M | 500 mM |

* All solutions mentioned above were prepared using 1× TBS buffer, pH 8.0.
* The sample was passed through the column at least 5 times, followed by washing with 20 CV of Equilibration Buffer.
* Elution Buffers I, II, III, and IV were applied sequentially, each using 5 CV. The eluates containing the target protein were collected.
* All collected fractions were stored frozen at -20°C.

## IX. Protein Concentration

### Material

Constant temperature water bath, centrifuge, micropipettes, 15 mL centrifuge tubes, 1.5 mL microcentrifuge tubes, vacuum freeze-dryer, purified protein solution, saturated ammonium sulfate solution, 1× TBS buffer.

### Protocol

#### Water Bath Concentration

The purified protein solution was placed in a preheated thermostatic water bath at 40°C. The tube caps were opened, and the tubes were tilted and heated until the remaining volume in each tube was approximately 1.5 mL.

#### Salting-out Concentration

* For each 12 mL tube of purified protein solution, slowly add a volume of saturated ammonium sulfate solution corresponding to a 3:2 ratio (ammonium sulfate solution : protein solution) with gentle mixing, and repeat this process. Incubate the mixture on an ice bath for 30 minutes.
* Aliquot the solution into 1.5 mL centrifuge tubes. Centrifuge at 4°C, 10,000 rpm for 10-15 minutes. Carefully discard the supernatant.
* Add 1-3 mL of saturated ammonium sulfate solution to resuspend the pellet. Centrifuge again at 4°C, 10,000 rpm for 5 minutes.
* Discard the supernatant, and resuspend the final pellet in 1× TBS Solution.

#### Lyophilization Concentration

* The caps of each tube containing the purified protein solution were loosened, the openings were covered with punctured paraffin film.
* The tubes were then placed in a pre-cooled vacuum freeze-dryer. The vacuum pump was activated, and lyophilization concentration was performed under conditions of -40°C and a vacuum pressure of 500 Pa for 10-12 hours.

## X. Protein Concentration Measurement

### Material

Thermostatic incubator (37°C), microplate reader, BCA protein assay kit, test samples.

### Protocol

#### Prepare the BSA Standard Sample

* Serially dilute the BSA standard (2 mg/mL) to generate standard curve samples with final concentrations as specified in the table below.

|  |  |
| --- | --- |
| Number | BSA Concentration (μg/mL) |
| A | 2000 |
| B | 1500 |
| C | 1000 |
| D | 750 |
| E | 500 |
| F | 250 |
| G | 125 |
| H | 25 |
| I | 0 |

* Prepare the BCA working reagent by thoroughly mixing BCA Reagent A and BCA Reagent B at a 50:1 volume ratio.
* Pipette 10 μL of each standard and unknown sample into individual wells of a 96-well plate. Subsequently, add 200 μL of the prepared BCA working reagent to each well. Incubate the plate at 37°C for 30 minutes, protected from light.
* Cool the plate to room temperature. Measure the absorbance of each well at a wavelength of 562 nm using a microplate reader. Perform blank subtraction on the acquired data.
* Generate a standard curve by plotting the blank-corrected mean absorbance values at 562 nm of the BSA standards against their corresponding concentrations (μg/mL).
* Determine the protein concentration of the samples by interpolating their blank-corrected absorbance values from the standard curve.

## XI. SDS-PAGE

### Material

Constant temperature water bath, Orbital shaker, Vertical slab electrophoresis system, Gel imaging and analysis system, DC regulated power supply, Gel casting cassette, Micropipettes, Tris-base, Glycine, SDS, SDS-PAGE gel preparation kit, Isopropanol, Protein loading buffer, Coomassie Brilliant Blue R250 staining solution (0.25 g Coomassie R250, 45 mL methanol, 10 mL glacial acetic acid, 45 mL deionized water), Gel destaining solution (250 mL methanol, 80 mL glacial acetic acid, brought to 1 L with deionized water), Test samples, Standard protein marker, Deionized water.

### Protocol

#### Sample Treatment

Combine the protein samples with 5× protein loading buffer at a ratio of 3:1 (sample : buffer). Denature the mixtures by heating at 100°C for 5 minutes in a heating block or water bath, then cool them to room temperature prior to loading.

#### Gel Preparation

Prepare the separating gel solution according to the formulations listed in the table below. Immediately after preparation, pipette the solution between the fixed glass plates and overlay with a layer of isopropanol to ensure a flat interface. After complete polymerization of the separating gel, pour off the isopropanol layer. Subsequently, prepare the stacking gel solution as specified in the table and promptly introduce it into the assembly. Carefully insert a comb into the stacking gel solution, avoiding air bubbles.

|  |  |  |
| --- | --- | --- |
| Component (Volume: mL) | 12% SDS-PAGE separating gel | SDS-PAGE stacking gel |
| Distilled Water | 1.000 | 1.4000 |
| 30% Acr-Bis Solution (29:1) | 2.000 | 0.330 |
| 1M Tris, pH 8.8 | 1.900 | 0.250 |
| 10%SDS | 0.050 | 0.020 |
| 10% Ammonium Persulfate (APS) | 0.050 | 0.020 |
| TEMED Substitute | 0.002 | 0.002 |
| Total | 5.000 | 2.000 |

Prepare 1x electrophoresis running buffer according to the composition specified in the table below.

|  |  |
| --- | --- |
| Component | Concentration |
| Tris | 3.029 g/L |
| Glycine | 14.413 g/L |
| SDS | 1 g/L |

* Secure the pre-cast gel within the electrophoresis unit, fill the buffer chambers with the prepared running buffer, remove the comb.
* Load the standard samples and test samples into the wells in sequence. Perform electrophoresis initially at 80 V for 30 minutes, until the samples are concentrated at the top of the separating gel. Then, increase the voltage to 130 V and continue electrophoresis for 40 minutes. Remove the gel and discard the stacking gel portion.
* Immerse the separating gel portion of the above gel in Coomassie Brilliant Blue R250 staining solution. Stain with constant shaking for 45 minutes.
* Transfer the stained gel into a container with destaining solution and agitate for 30 minutes, then replace the destaining solution. Repeat the destaining process 4-5 times. Observe and document the results using a gel imaging analysis system.

## XII. Protein Characterization Experiments

### MTT Assay for Cell Viability

#### Protocol

* Cells were seeded in 96-well plates at a density of 6,000 cells per well and cultured for 24 hours in a humidified atmosphere at 37°C with 5% CO₂ to allow cell adhesion.
* The original medium was removed and replaced with fresh medium containing varying concentrations of the TRACER protein. A blank control group containing only medium (without cells) was also established.
* After 48 hours of treatment, 10 μL of MTT solution (5 mg/mL in PBS) was added to each well, followed by incubation at 37°C protected from light for an additional 3.5 hours.
* The supernatant was carefully aspirated, and the resulting formazan crystals were dissolved in 100 μL of DMSO. The plates were subjected to gentle shaking for 10 minutes to ensure complete dissolution. The absorbance was measured at a wavelength of 570 nm using a multi-function microplate reader.
* Cell viability was calculated as follows:

Cell viability (%) = [(ODexperiment − ODblank) / (ODcontrol − ODblank)] × 100%

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![Results banner](https://static.igem.wiki/teams/5623/static/images/intro/result.avif)

# Results

"From Concept to Reality: Our Experimental Journey"

* [Introduction](#introduction)
* [Result 1](#result1)
* [Result 2](#result2)
* [Result 3](#result3)

## Introduction

Our experimental objective is to produce the designed fusion protein using *Escherichia coli* BL21(DE3) and characterize the function of the deliver. **We successfully constructed a plasmid with the fusion protein gene and attempted to induce the extraction of the fusion protein.**

## Result 1: Gel Electrophoresis Verification

##### Gel electrophoresis of recombinant plasmid DNA for verification of digestion

We constructed pET-28a(+)-TRACER-IL24 recombinant plasmids and pUC19-TRACER-IL24 recombinant plasmids as shown in Fig.1a. and Fig.2a.. The nucleic acid electrophoresis bands after double enzyme digestion were consistent with expectations, **confirming that the insertion was correct.**

As shown in Fig.1b., Lane 1 is Marker III; Lane 2 is a complete pET-28a(+) with a theoretical length of 5369bp; Lanes 3-4 show the double-digested pET-28a(+) vector, with a theoretical length of 5350 bp; Lanes 5-6 display the PCR-amplified target fragment, with a theoretical length of 697 bp. **All results match the expected lengths.**

![Figure 1: Recombinant Plasmid pET-28a](https://static.igem.wiki/teams/5623/static/images/results/figure1.avif)

**Fig 1.** Illustration of the recombinant plasmid. A. pET-28a(+)-TRACER-IL24 recombinant plasmid map. B. Nucleic acid electrophoresis is used to verify the enzymatic digestion results.

![Figure 2: Recombinant Plasmid pUC19](https://static.igem.wiki/teams/5623/static/images/results/figure2.avif)

**Fig 2.** Illustration of the recombinant plasmid. A. Map of the recombinant plasmid pUC19-TRACER-IL24. B. Nucleic acid electrophoresis was used to verify the enzymatic digestion results.

## Result 2: Protein Purification Analysis

##### Protein purification, determination of protein concentration by BCA method, and characterization of protein molecular weight by SDS-PAGE

We attempted to extract and purify the target protein from the bacterial culture, but **the initial protein concentration was low, and no distinct bands were observed in the SDS-PAGE analysis, rendering it unsuitable for subsequent experimental requirements.**

###### Approach 1: Direct Purification

The standard curve determined and plotted using the BCA method is shown in Fig 3. In SDS-PAGE, **no distinct bands were observed except for the protein marker** (Fig 4).

The results of the BCA method and SDS-PAGE experiments indicated that **the extracted protein concentration was relatively low, and no obvious bands could appear on the SDS-PAGE,** suggesting that **the protein concentration we extracted could not meet the requirements of subsequent experiments.**

![Figure 3: BCA Method Results](https://static.igem.wiki/teams/5623/static/images/results/figure3.avif)

**Fig 3.** Results of protein concentration determination by BCA method

![Figure 4: SDS-PAGE Results](https://static.igem.wiki/teams/5623/static/images/results/figure4.avif)

**Fig 4.** SDS-PAGE characterization results of protein molecular weight

###### Approach 2: Enhanced Induction and Concentration

Firstly, **increasing the induced concentration of IPTG and using three concentration methods** (water bath concentration, ammonium sulfate precipitation concentration, and freeze-drying concentration) **did not achieve the expected results**.

The standard curve determined and plotted using the BCA method is shown in Fig 5. Calculate the corresponding concentrations using the assay data. Among the results obtained from the three protein extraction methods, freeze-dried concentration yielded the best concentration results. However, **the protein obtained using this method still failed to meet our requirements**.

The results calculated based on the BCA method indicated that **even after enhanced induction and the application of three concentration methods respectively, the protein concentration did not show a significant increase.**

![Figure 5: Enhanced BCA Results](https://static.igem.wiki/teams/5623/static/images/results/figure5.avif)

**Fig 5.** Results of protein concentration determination by BCA method - Enhanced induction

###### Approach 3: Extracting Fusion Proteins Through Precipitation

![Figure 6: TRACER Solubility Analysis](https://static.igem.wiki/teams/5623/static/images/results/figure6.avif)

**Fig 6.** TRACER protein solubility analysis

Subsequently, we attempted to **extract the fusion protein from the ultrasonication-treated pellet, but the concentration remained insufficient for subsequent experiments** (Table 1).

Based on the modeling group results (Fig 6), the information indicates that **the fusion protein has low solubility and may be present in the precipitate. So we decided to extract the proteins from the supernatant and the precipitate respectively after ultrasonic disruption.**

We determined the protein concentration by the BCA method and plotted the standard curves (Fig 7a and 7b). Analysis of the measurement data indicates that **although the protein concentration of the purified sample has significantly increased, the final value still falls short of the level required for subsequent experiments**. This clearly indicates that **our current protein extraction and concentration processes have bottlenecks and need to be optimized**.

![Figure 7a: BCA Results First Experiment](https://static.igem.wiki/teams/5623/static/images/results/figure7a.avif)

![Figure 7b: BCA Results Second Experiment](https://static.igem.wiki/teams/5623/static/images/results/figure7b.avif)

**Fig 7.** Results of protein concentration determination by BCA method. A. Results of the first experiment. B. Results of the second experiment.

**Table 1.** Results of Protein concentration Determination by BCA Method

| Purification Process | Maximum concentration of purified samples | Note |
| --- | --- | --- |
| Direct Purification | 179.4 μg/ml | - |
| Enhance the intensity of induced expression and perform concentration | 114.7 μg/ml | Excessive freeze-thaw cycles during the concentration process led to reduced protein concentration, resulting in ineffective concentration. |
| Extraction of fusion protein from supernatant and precipitate | 247.6 μg/ml | Neither the supernatant nor the precipitate exhibited elevated protein concentrations, and the highest concentration of purified sample was found in the supernatant. |

## Result 3: Engineered Bacteria as Smart Carriers

##### Escherichia coli was used as the factory and drug delivery vector

In this system, engineered bacteria are delivered directly into the body. The core mechanism lies in: the sustainable expression of fusion proteins under the control of the constitutive promoter Pj23100; When bacteria target colorectal cancer tissues, they activate specific lysis programs, thereby releasing therapeutic proteins locally at the lesion site and achieving **precise drug delivery.**

![Figure 10: Redesigned Genetic Circuit](https://static.igem.wiki/teams/5623/static/images/results/figure10.avif)

**Fig 8.** The redesigned genetic circuit

**Note:** Due to the submission deadline of iGEM Wiki, we have not yet completed the subsequent characterization experiments and plan to report the relevant experimental results in the presentation session of Jamboree.

###### NetLogo Simulation Analysis

Additionally, we employed NetLogo to conduct organizational-level simulations, analyzing the relationship between inoculation levels and lysis cycles, and obtained corresponding spatiotemporal distribution visualization data.

As shown in Fig 9, different initial inoculation amounts have a significant impact on biomass accumulation and pyrolysis cycle: with the increase of inoculation amount, the pyrolysis cycle is significantly shortened, and the population synchrony is enhanced. Simulation results indicate that **the initial inoculum size regulates the lysis cycle of the entire population by determining the accumulation rate of the quorum-sensing signal.**

![Figure 11: Biomass and Lysis Cycle Analysis](https://static.igem.wiki/teams/5623/static/images/engineering/figure20.avif)

**Fig 9.** Effects of different initial inoculum sizes on lysis cycle

The spatiotemporal visualization results from the NetLogo simulation (Fig 10) reveal that **higher inoculation doses lead to a more concentrated spatiotemporal distribution of lysis events and exhibit a more stable periodic pattern.**

![Figure 12: Spatiotemporal Visualization](https://static.igem.wiki/teams/5623/static/images/results/figure12.avif)

**Fig 10.** Spatiotemporal visualization results of NetLogo simulation

×
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