iGEM 2024 — Team jlu-china

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## **Month**

[April](#part1)
[May](#part2)
[June](#part3)
[July](#part4)
[August](#part5)
[September](#part6)

## **Notebook**

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### **Introduction**

Our project started from April to September, focusing on the development and assessment of bionic fluorescent proteins labeled with near-infrared type-II (NIR-II) dyes for advanced biomedical imaging. The research was meticulously planned and executed over several months, beginning with foundational laboratory training and culminating in the establishment of mouse models for in vivo imaging.

### **April**

**Week 1: Laboratory Access Training and Assessment**

This week, we participated in safety training at the Translational Medicine Research Institute of Jilin University, covering access qualifications for cell rooms, animal facilities, and public instrument platforms. The training encompassed laboratory safety regulations, animal experimentation procedures, rules for entering and exiting the animal facilities, and approaches to handling major safety issues.

**Weeks 2 & 3: Basic Experimental Skills Training**

During this period, we gained familiarity with the laboratory's equipment and fundamental experimental operations, including cell culture, bacterial culture, and the utilization of instruments such as shakers, incubators, microplate readers, and UV-Vis spectrophotometers. We had our first attempt at Escherichia coli cultivation.

**Week 4: Experimental Design and Discussion in Detail**

Collaborating with our supervising instructor, we deliberated on the experimental plan and its specifics, clarifying the primary objectives and anticipated outcomes. We formulated a detailed timeline, outlining the time nodes and critical tasks for each experimental phase.

### **May: constructing bionic fluorescent proteins**

**Weeks 1 & 2: Screening of Protein-Labeling NIR-II Dyes**

Given the current lack of NIR-II dyes that can directly and covalently label proteins, we embarked on a screening process involving various common NIR-II dyes such as IR-26, IR-1061, Et-1080, and CO-1080. These dyes were co-incubated with potential protein carriers in vitro. By adjusting factors like reaction time, temperature, and feed ratio, we successfully identified that CO-1080 could bind relatively quickly with human serum albumin under mild conditions.

**Week 3: Exploring Structures and Binding Capabilities**

To gain deeper insights into why different dye molecules exhibit varying binding efficiencies with proteins, we attempted to uncover the relationship between molecular structures and their affinity towards proteins using molecular docking methods.

**Week 4: Quantifying Binding Capabilities**

To accurately assess the construction efficiency of biomimetic fluorescent proteins, we employed protein electrophoresis and high-resolution mass spectrometry to isolate and determine the precise proportion of the protein-bound NIR-II luminescent core (CO-1080) within the mixture before and after binding. The results indicated that although the protein could be almost completely labeled by the dye under heated conditions, further optimization was necessary to reduce the reaction temperature and time, which were higher and longer than those encountered in living organisms.

### **June: Identification of Binding Sites**

**Weeks 1 & 2: Confirmation of Dye-Protein Binding Sites**

Based on previous reports, albumin is known to have three structural domains. We obtained recombinant proteins of these three domains and incubated them with the dye in vitro, observing their brightness under an NIR-II camera. The results revealed that one particular domain significantly enhanced the dye's fluorescence. Additionally, gel electrophoresis indicated that this domain could fully bind with the dye to form a stable complex.

**Weeks 3 & 4: Proteomic Identification of Specific Binding Sites**

Proteomic analysis was performed on the resulting biomimetic fluorescent proteins using Nano LC-MS/MS to identify the specific amino acid residue sites where the dye was bound.

### **July: Preparation of Mutant Proteins**

**Week 1: Design of Mutant Protein Sequences**

scores, consistent with our prediction that the reaction between the dye and protein occurs at the thiol groups of cysteines. However, we also noted that these sites primarily form disulfide bonds within the protein structure. After discussing with our supervisor, we planned to mutate these amino acid sites to achieve more efficient protein-dye binding.

**Weeks 2 & 3: Confirmation of Experimental Feasibility and Preparation**

This week, we purchased the necessary reagents and consumables, learned the relevant protein expression techniques, and initiated experiments for protein expression.

**Week 4: Continued Optimization of Mutant Protein Preparation and Successful Expression of Small Quantities**

We successfully obtained small quantities of mutant proteins and co-incubated them with the dye. The addition of mutant proteins accelerated the reaction rate between the dye and protein, enabling the preparation of biomimetic fluorescent proteins at 37°C.

### **August: Assessment of Dye Biocompatibility and In Vivo Imaging Capabilities**

**Weeks 1 & 2: Biocompatibility Assessment**

Materials intended for direct use in biological systems must undergo thorough biocompatibility assessments. This week, we cultured several types of common cells, including normal tissue cells and cancer cells. MTT assays were performed to verify whether the biomimetic fluorescent proteins had acceptable toxicity levels towards these cells. Subsequently, the proteins were injected into mice, and their acute and chronic toxicity within the animals was evaluated through blood tests (routine, liver function, kidney function), and histopathological examinations of major organs.

**Week 3: Systematic Evaluation of Metabolic Capabilities Considering Potential Clinical Applications**

We assessed the metabolic profiles of different dyes, determining the blood half-lives of the biomimetic fluorescent proteins and the organ distribution of their fluorescent signals.

**Weeks 4: In Vivo Imaging Capabilities Assessment**

After administering the biomimetic fluorescent proteins through footpad or tail vein injection, mice were immediately imaged using an NIR-II camera for visualization of blood vessels and lymphatic vessels. Imaging capabilities were evaluated by quantifying light-up times and signal-to-noise ratios.

### **September: Mouse Model Establishment and Imaging**

**Week 1: Mouse Model Construction**

We experimented with varying laser irradiation durations to determine the optimal level of barrier disruption for achieving the best imaging outcomes and detection thresholds.

**Weeks 2 & 3: In Vivo Imaging**

Following model establishment, mice were injected with different biomimetic fluorescent proteins and unmodified free dyes. Under NIR-II imaging, the fluorescent signals in the modeled regions were continuously observed and quantified. After the experiments, mice were euthanized, and tissues were harvested for quantitative analysis of fluorescent leakage in both damaged and normal tissues.

**Week 4: Data Compilation and Analysis**

All data were compiled, and their relevance and reliability were verified.

### **Weekly Notebook**

Unable to display PDF file. [Download](https://static.igem.wiki/teams/5209/images/notebook/weekly-notebook.pdf) instead.

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
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## **Menu**

**Problem Definition**

[Issues with the Probe Molecule](#part1.1)
[Preliminary Solution](#part1.2)

**Protein Engineering Design Iteration and Validation**

[Objective](#part2.1)
[CO-1080 and HSA Binding Analysis](#part2.2)
[Mutation Strategy](#part2.3)
[Experimental Validation](#part2.4)
[Optimization and Further Exploration](#part2.5)

## **Engineering**

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### **Problem Definition**

##### Issues with the Probe Molecule:

When used independently, NIR-II probes such as CO-1080 face several challenges that significantly limit their effectiveness for in vivo imaging. One of the primary issues is their rapid degradation within biological environments, which reduces the probe's functional lifespan and makes long-term imaging difficult. Additionally, the probes are prone to being quickly cleared from the body by the liver and kidneys, resulting in short circulation times. Another major challenge is the formation of a protein corona—a layer of proteins that adsorbs onto the surface of the probe upon its introduction into the bloodstream. This protein corona can alter the surface properties of the probe, impacting its ability to target specific tissues and diminishing its imaging efficiency. These limitations make it difficult for standalone NIR-II probes to perform effectively in complex biological systems, particularly for deep tissue imaging and long-duration studies.

##### Preliminary Solution:

To overcome these challenges, we propose encapsulating the NIR-II probe within human serum albumin (HSA). HSA is a highly biocompatible protein naturally present in the human body, and it serves as an ideal carrier to improve the in vivo behavior of the probe. By encapsulating the probe within HSA, we aim to enhance the probe’s stability, protecting it from rapid degradation.

Furthermore, the HSA coating can help prevent the probe from being rapidly cleared from circulation, thereby extending its half-life and allowing it to remain in the bloodstream for longer periods. The HSA encapsulation also acts as a protective barrier against the formation of a protein corona, preserving the probe's surface properties and maintaining its targeting efficiency. Ultimately, this step is expected to significantly improve the biocompatibility, circulation time, and overall imaging performance of the probe, enabling more reliable and effective in vivo imaging applications, especially in complex biological environments.

![engineering1](https://static.igem.wiki/teams/5209/images/engineering/engineering-pic1.webp)

Fluorescence spectra of CO-1080 in DMSO solution, CO-1080 in PBS solution, and HSA@CO-1080 FPs under 1064 nm excitation

![engineering2](https://static.igem.wiki/teams/5209/images/engineering/engineering-pic2.webp)

Photostability of CO-1080 and HSA@CO-1080 FPs under 1064 nm excitation (mean ± SD, n = 3 independent samples per group).

### **Protein Engineering Design Iteration and Validation**

#### **Objective:**

Building on the encapsulation of the CO-1080 NIR-II probe with HSA, we sought to further investigate the molecular interactions between CO-1080 and HSA to enhance their binding efficiency. The ultimate goal was to improve the performance of the probe in biological environments, particularly in NIR-II imaging applications, by optimizing the binding affinity between CO-1080 and HSA through site-directed mutagenesis. This iterative design process aims to create a more robust system, where enhanced protein-probe interactions improve the probes' stability and effectiveness for bioimaging.

#### **CO-1080 and HSA Binding Analysis:**

![engineering3](https://static.igem.wiki/teams/5209/images/engineering/engineering-pic3.webp)

Previous studies have suggested that CO-1080 tends to covalently bind to domain III (DIII) of HSA. Proteomic analysis identified Cys477 and Cys487 as potential covalent binding sites for CO-1080. Among these, Cys477 showed a higher binding score, making it the most likely covalent binding site for CO-1080. The presence of reversible disulfide bonds between Cys476 and Cys487, as well as between Cys461 and Cys477, provided an opportunity to explore mutations that could break these bonds, release free –SH groups, and potentially accelerate the binding of CO-1080 to HSA.

![engineering4](https://static.igem.wiki/teams/5209/images/engineering/engineering-pic4.webp)

gel electrophoresis analysis (n = 4 independent experiment) of the binding ability between CO-1080 chromophores and HSA, DI, DII, DIII.

To determine whether such mutations would affect the non-covalent affinity between CO-1080 and HSA, we performed molecular docking simulations using the Glide software. These simulations allowed us to predict the non-covalent interactions between CO1080 and recombinant HSA. The mutation of Cys476 to Gly (C476G) was introduced, and docking scores and binding energies were calculated using the MM/GBSA method. Results demonstrated that all single cysteine mutations enhanced the non-covalent binding affinity of recombinant HSA to CO-1080, with the Cys476G mutation showing the most significant improvement.

#### **Mutation Strategy:**

Based on the results of the molecular docking simulations, we focused on site-directed mutagenesis of key cysteine residues in HSA, specifically Cys476 and Cys477, to enhance CO-1080 binding efficiency. The hypothesis was that breaking the disulfide bonds between Cys476 and Cys477, or Cys461 and Cys487, would free –SH groups that could enhance covalent binding with CO1080. The primary mutation strategy involved replacing Cys476 with Gly (C476G), which was predicted to expand the protein cavity, expose more binding sites, and increase the overall affinity between the probe and the protein.

![engineering5](https://static.igem.wiki/teams/5209/images/engineering/engineering-pic5.webp)

#### **Experimental Validation:**

Following the docking simulations, we produced recombinant HSA (rHSA) with the Cys476-to-Gly mutation using site-directed mutagenesis (SDM). Experimental validation confirmed that the C476G mutation led to an expansion of the protein cavity, which facilitated easier access of CO-1080 to the binding site. The mutation also exposed free –SH groups that accelerated the covalent binding reaction between CO-1080 and rHSA, particularly under mild conditions at room temperature. These findings confirmed the success of the C476G mutation in improving both non-covalent and covalent binding interactions.

![engineering6](https://static.igem.wiki/teams/5209/images/eg/engineering-pic6.webp)

Gel electrophoresis results of CO-1080@albumin incubated at room temperature for 2 h

#### **Optimization and Further Exploration:**

Further validation was performed using electrophoresis and mass spectrometry to quantify the binding efficiency of the mutated rHSA. The results demonstrated significant improvements in both non-covalent and covalent binding efficiency for the C476G mutation compared to wild-type HSA. Moving forward, we plan to use GaMD (Gaussian Accelerated Molecular Dynamics) or meta dynamics simulations to explore the binding pathway of the CO-1080 probe molecule into the HSA binding site.

These simulation techniques will help identify key residues involved in the binding pathway and provide detailed insights into their roles during the binding process. We will also investigate the spatial and electronic effects of nearby residues along the binding pathway, assessing their potential impact on binding efficiency. Through this analysis, we aim to further optimize the interaction between the probe and protein, ultimately improving the binding efficiency of the CO-1080@rHSA complex and enhancing its potential for NIR-II bioimaging applications

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## **Menu**

[Engineering Success](#part1)
[Imaging Challenges: Blood-Testis Barrier (BTB) and Blood-Brain Barrier (BBB)](#part2)
[BTB Imaging Results](#part3)
[BBB Imaging Results](#part4)
[Conclusion](#part5)

## **Results**

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### **Engineering Success**

Through multiple rounds of design iteration, we successfully developed a recombinant human serum albumin C476G mutant (rHSA-C476G), significantly enhancing the binding efficiency with the NIR-II dye CO-1080. By introducing a site-directed mutation at Cys476 (replacing Cys476 with Gly476), we disrupted the original disulfide bond and exposed a free –SH group, thereby improving the covalent binding between the protein and CO-1080. This mutation strategy not only optimized the binding site but also expanded the protein cavity, further enhancing the probe’s access to the binding site.

**Re\_Fig1A** shows the molecular mechanism of covalent binding between CO-1080 and the rHSA-C476G mutant. After the mutation, the disulfide bond is broken, exposing the –SH group of Cys476, which facilitates the reaction with CO-1080. The optimization of the binding site is further validated through molecular docking simulations (**Re\_Fig1B**), where the results clearly show how CO-1080 binds more tightly to the active site of the rHSA-C476G mutant, with significantly enhanced non-covalent affinity. The binding energy calculations (**Re\_Fig1C and 1D**) demonstrate that the binding energy of rHSA-C476G is far superior to that of wild-type HSA and other mutant groups, indicating that the C476G mutation significantly improves binding efficiency and stability.

![results1](https://static.igem.wiki/teams/5209/images/results/results-pic1.webp)

Re\_Fig1: **Mutant albumin with better binding property to the NIR-II chromophore.** (A) Schematic of the disulfide bonds between the cysteines associated with the covalent binding of CO-1080 and HSA. rHSA stands for recombinant HSA and C476G indicates mutation of Cys476 to Gly476. Cys: cysteine, Gly: glycine (B) Theoretical simulation of HSA and rHSA-C476G binding to CO-1080 by gliding docking mode. Comparison of (C) docking score and (D) binding energy of CO-1080 to wild-type HSA and several groups of recombinant HSA, respectively. (E) NIR-II brightness of CO-1080@albumin under different conditions (mean ± SD, n=3 for each group). (F) Gel electrophoresis results of CO-1080@albumin incubated at room temperature for 2 h and (G) statistics on the ratio of CO-1080@albumin to the corresponding free dye

Experimental results further support these findings. In the comparison of fluorescence intensity (**Re\_Fig 5E**), the NIR-II fluorescence signal of CO-1080 bound to the rHSA-C476G mutant is much higher than that of other mutants and wild-type HSA. This indicates that the C476G mutation not only enhances the binding efficiency of the probe but also improves the imaging performance of the fluorescent probe. Gel electrophoresis analysis (**Re\_Fig 5F**) further confirms that the binding capacity of rHSA-C476G to CO-1080 is significantly higher than that of other mutants and wild-type HSA after incubation at room temperature for 2 hours. The statistical results of bound versus free dye (**Re\_Fig 5G**) show that rHSA-C476G has the highest efficiency in binding CO-1080, with the lowest ratio of free dye, demonstrating that the mutant efficiently bound more probe molecules in a shorter time.

Additionally, **Re\_Fig 2A** illustrates the process of constructing the rHSA-C476G mutant. Through site-directed mutagenesis and expression in a yeast system, we successfully obtained the recombinant HSA mutant. This mutation not only optimized the binding site for CO-1080 but also transformed the mutated protein cavity into an efficient microreactor that could covalently bind the probe under mild conditions without requiring additional heating steps. The figure clearly depicts how the rHSA-C476G mutant improves the interaction with CO-1080 without altering the overall structure of the protein.

![results2](https://static.igem.wiki/teams/5209/images/results/results-pic2.webp)

Re\_Fig2：Optimized recombinant HSA constructed by site-directed mutagenesis strategy

Overall, experimental validation confirmed the effectiveness of this design. The optimized rHSA-C476G mutant significantly improved both the non-covalent affinity and covalent binding ability with CO-1080, forming an efficient biomimetic fluorescent protein probe. This optimized probe is not only suitable for NIR-II imaging but also provides a stable and efficient tool for **non-invasive deep tissue imaging**, offering broad potential for future applications.

### **Imaging Challenges: Blood-Testis Barrier (BTB) and Blood-Brain Barrier (BBB)**

Both the blood-testis barrier (BTB) and blood-brain barrier (BBB) present unique challenges for imaging due to their complex structures and selective permeability. These biological barriers are essential for maintaining the microenvironment of sensitive organs such as the brain and testes, but their disruption is difficult to assess using traditional imaging techniques.

The BTB is formed by tight junctions between Sertoli cells in the testes, which protect germ cells from toxins and immune responses. However, when the BTB is compromised due to trauma, infection, or disease, it becomes challenging to evaluate the extent of the damage because of the barrier’s deep location and selective permeability. Traditional methods such as tissue staining or contrast-based imaging often fail to provide real-time, non-invasive assessments of barrier integrity. Moreover, these techniques typically lack sufficient resolution and specificity, making it difficult to distinguish between healthy and damaged tissue, particularly in deep areas like the testes. This can lead to inaccurate diagnoses or delays in treatment.

![results3](https://static.igem.wiki/teams/5209/images/results/results-pic3.webp)

Re\_Fig3: Schematic of the in situ formed dye@rHSA FPs penetrating through the damaged BTB and accumulating in the testes

Similarly, the BBB serves as a protective interface between the central nervous system and the bloodstream, preventing harmful substances from entering the brain while allowing essential nutrients to pass through. Disruption of the BBB is often associated with severe neurological conditions, such as stroke, traumatic brain injury, and neurodegenerative diseases. However, traditional imaging techniques face similar challenges when assessing the BBB, especially in detecting early-stage damage. Conventional methods often lack the sensitivity needed to precisely visualize damaged areas, further complicating the evaluation of BBB integrity.

![results4](https://static.igem.wiki/teams/5209/images/results/results-pic4.webp)

Re\_Fig4: Targeted imaging of blood-brain barrier (BBB) disruption using the in situ formed dye@rHSA FPs

Given these challenges, we sought to test our engineered fluorescent protein probe in these difficult-to-image environments. The unique properties of our probe, including its enhanced binding efficiency and strong NIR-II fluorescence, make it an ideal candidate for assessing the integrity of both the BTB and BBB.

### **BTB Imaging Results**

#### **Experimental Validation:**

The experimental results demonstrated that CO-1080@rHSA exhibited superior imaging capabilities in BTB-damaged mice, showing significantly higher brightness and testis-to-skin ratio (TSR) compared to the control groups. **Re\_Fig5 B and C** show the NIR-II whole-body and testicular imaging of BTB-damaged and normal mice after the injection of CO-1080@rHSA and CO-1080@HSA. The images indicate that CO-1080@rHSA allowed for clearer differentiation between normal and damaged testes, starting as early as 3 hours post-injection and persisting up to 72 hours.

![results5](https://static.igem.wiki/teams/5209/images/results/results-pic5.webp)

Re\_Fig5: NIR-II Imaging of BTB Integrity Using CO-1080@rHSA

**Re\_Fig5 D** highlights the brightness measurements of the testes and skin in the treated and control groups, with CO-1080@rHSA achieving a higher fluorescence intensity, particularly in the BTB-damaged group. **Re\_Fig5 E** provides statistical analysis, demonstrating a significant difference in testis-to-skin ratios between the CO-1080@rHSA and CO-1080@HSA groups at 6 hours post-injection, validating the designed probe's efficacy in complex biological environments.

These imaging results not only provide a new method for real-time, non-invasive assessment of BTB integrity but also offer potential support in diagnosing conditions related to male infertility. Future research could focus on longitudinal monitoring of BTB integrity to assess the progression of testicular pathologies and the effectiveness of therapeutic interventions.

### **BBB Imaging Results**

In this study, the probes used were not CO-1080 but other molecules, such as C7-1080 and IR-808Ac, which were primarily used to detect blood-brain barrier (BBB) disruption in a stroke model. C7-1080 is a near-infrared II (NIR-II) dye that binds to recombinant HSA mutant (rHSA) and provides clear imaging of rHSA infiltration into brain tissue in the stroke model through NIR-II imaging. In contrast, IR-808Ac was mainly used for vascular imaging to observe blood flow around the stroke area.

As shown in the figures, **Re\_Fig6 A** demonstrates the imaging results of C7-1080 and IR-808Ac from 1 minute to 48 hours. The C7-1080 probe shows progressively stronger signals in the stroke region, clearly illustrating BBB leakage. In comparison, IR-808Ac produces weaker signals, primarily for vascular imaging. **Re\_Fig6 B** provides a quantitative analysis of C7-1080 fluorescence intensity at different time points, revealing significantly stronger signals in the stroke model compared to the control group (Sham), particularly in the early detection phase (1 minute to 6 hours), indicating C7-1080's ability to detect early BBB disruption. **Re\_Fig6 C** compares traditional staining methods (TTC and EB) with NIR-II imaging, showing that C7-1080 provides clearer and more precise visualization of BBB disruption, with superior resolution and sensitivity compared to traditional techniques. **Re\_Fig6 D**  further validates the high sensitivity of C7-1080, showing significantly higher fluorescence in the affected brain hemisphere in the stroke model compared to the Sham group. **Re\_Fig6 E** displays the absorption spectra of C7-1080 and IR-808Ac, with C7-1080 having strong absorption at 1046 nm, making it ideal for NIR-II imaging, while IR-808Ac has stronger absorption at 796 nm, suitable for NIR-I imaging. **Re\_Fig6 F** shows dual-channel imaging results at 40 minutes and 48 hours, where the red channel represents the C7-1080 signal and the green channel represents the IR-808Ac signal. This dual-channel imaging provides comprehensive information on the stroke area, arteries, and veins, simultaneously revealing both BBB disruption and vascular structure.

![results6](https://static.igem.wiki/teams/5209/images/results/results-pic6.webp)

Re\_Fig6: Detection of blood-brain barrier (BBB) disruption in a stroke model using C7-1080 and IR-808Ac @rHSA FPs.

C7-1080 demonstrated outstanding sensitivity and specificity in detecting BBB disruption, offering higher resolution compared to traditional staining methods. Additionally, dual-channel imaging combining C7-1080 and IR-808Ac allows for detailed visualization of both BBB disruption and vascular structure, further enhancing the imaging of the stroke area. These results confirm the high potential of C7-1080 in complex biological imaging applications.

### **Conclusion**

The engineered rHSA-C476G protein probe significantly enhanced covalent binding with the NIR-II dye CO-1080, establishing a robust platform for bioimaging applications. Both BTB and BBB imaging results confirmed the probe's effectiveness in overcoming complex imaging challenges, such as detecting early-stage disruption in biological barriers. The high sensitivity and specificity of the probe in non-invasive imaging provide promising potential for broader clinical applications, especially in the real-time monitoring of tissue integrity in sensitive environments.

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