iGEM 2024 — Team cnpem-brazil

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Welcome to the B.A.R.B.I.E Project’s Wet Lab Notebook! This document meticulously chronicles all the experimental techniques, findings, and analyses conducted by our team. It outlines the project’s key stages, covering each cycle of investigation, including bacterial transformations, protein expression, purification procedures, and more. This notebook offers insights into the methodologies we employed, the challenges we faced, and the breakthroughs we achieved throughout our iGEM project. We hope you enjoy exploring our scientific journey!

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You can download the PDF [here](https://static.igem.wiki/teams/5396/notebookpdf/notebookfinal.pdf).

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## Capture Module

#### Design

Last year, the CNPEM-BRAZIL team participated in the iGEM Design League, the Latin American spin-off of the iGEM competition. Our project, B.A.R.B.I.E. (**B**ioengineered **A**pproach for **R**emoval of **M**icroplastics through **B**ioremediation and **I**nnovative **E**lectromagnetics), was inspired by the Kyoto 2019 team, which used plastic-binding proteins to address plastic pollution.

Our approach involved a bio-based system designed to detect and remove microplastics (MPs) from water treatment plants (WTPs). We combined encapsulin, a protein from *Quasibacillus thermotolerans* capable of mineralizing iron, with magnetic particles. This allowed us to create a magnetic complex capable of being pulled out by external magnetic fields. Attached to the encapsulin was BaCBM2, a plastic-binding protein that adheres to various plastics, and the SpyTag-SpyCatcher system to link the components.

The idea was to employ this magnetic protein complex (MBPE) to water treatment plants, where it would bind to MPs. Using magnetic fields, we could then remove the MPs along with the encapsulin complex. While innovative, this strategy proved too complex and hard to implement in real life, leading us to a completely new approach this year.

### Cycle 1

#### Design

Cycle 1 of B.A.R.B.I.E. 4.0 started this year, focusing on simplifying and improving the performance of plastic-binding proteins (PBPs) to enhance microplastic removal. Based on lessons learned from last year’s complex encapsulin-based system, we chose to streamline the approach by replacing encapsulin with Mad10, a small peptide that still allowed magnetic manipulation with a great reduction of the system's complexity. At this stage, our focus remained on water treatment plants (WTPs), as we had not yet transitioned to household filters.

A key part of our design was the inclusion of specific elements in the protein circuit: a 6xHis tag, the plastic-binding protein (PBP), miRFP (a red fluorescent protein), and three repeats of the Mad10 peptide.

* **6xHis tag:** This was added to simplify the protein purification process. By including a 6xHis tag, we ensured that the proteins could be easily purified using nickel affinity chromatography. This tag binds strongly to nickel ions, allowing us to isolate PBPs from other cellular proteins in a single purification step, enhancing the efficiency and speed of the protein production process.
* **PBP (Plastic-Binding Protein):** The core of the system is the PBPs (Bacillus subtilis BaCBM2 and our designed protein BARBIE1), which are responsible for binding to various types of plastics. The goal was to enhance BARBIE1 affinity to different plastic types through our design pipeline, ensuring that the protein could efficiently capture microplastics in solution.
* **miRFP (Red Fluorescent Protein):** We included miRFP as a visual marker to monitor the expression and localization of the protein. The red fluorescence would allow the tracking and confirm the presence of the protein in the system, ensuring that it was being correctly expressed and localized where expected.
* **3xMAD10:** The three repeats of the Mad10 peptide were included to retain the magnetic properties that were present in the original encapsulin design, but in a much simpler form. Mad10 is a peptide known to bind to magnetic particles, allowing the protein to be manipulated by an external magnetic field. By including three copies of Mad10, we aimed to increase the magnetic responsiveness of the system, enabling more efficient capture and removal of microplastics when combined with magnetic separation techniques.

In this cycle, we also developed a novel pipeline for designing PBPs with enhanced affinity for pollutants, especially microplastics. The steps involved in creating these engineered proteins included:

* Generating the structure of BaCBM2 using AlphaFold2.
* Performing docking assays with six common plastics (PP: Polypropylene, PE: Polyethylene, PET: Polyethylene Terephthalate, NY: Nylon, PVC: Polyvinyl Chloride and PS: Polystyrene) using Gnina software.
* Removing overlaps and visualizing sequences using ChimeraX.
* Using reverse folding with LigandMPNN to generate unique protein sequences.
* Retrieving the consensus sequence from the 36,000 designed proteins to identify structural features shared across all tested plastics. This consensus sequence led to the creation of our most optimized plastic-binding protein, BARBIE1.

BARBIE1 was then validated through additional docking assays, which confirmed its increased affinity for all plastics compared to the original BaCBM2, representing a significant advancement in our protein design capabilities.

#### Build

In this step, the focus was on building the newly designed plastic-binding protein, BARBIE1, generated from our protein design pipeline. The protein was derived from BaCBM2 and optimized for enhanced plastic affinity, as described in the design phase. To validate this new protein, we needed to ensure its successful expression, purification, and structural integrity.

We began by purchasing the DNA synthesis of the BARBIE1 sequence, which was the consensus from our pool of 36,000 designed proteins. The synthesized gene is cloned into the pET-15b expression vector, which utilizes the T7 promoter for high-level protein expression. This vector also included a 6xHis tag for ease of purification by affinity chromatography.

This construction was transformed it into *Escherichia coli* BL21(DE3), a strain commonly used for recombinant protein production due to its ability to express proteins under the control of the T7 promoter.

#### Test

After constructing the BARBIE1 protein, we conducted a series of in silico and experimental assays to evaluate its structural integrity, functionality, and plastic-binding efficiency, comparing it to BaCBM2.

***In Silico* Tests**

We began by using **AlphaFold 3** to predict BARBIE1's structural properties. The prediction indicated that BARBIE1 is likely to exist as a monomer, with a folding pattern highly similar to BaCBM2. This was crucial for ensuring that the modifications made during the design phase did not compromise the protein’s overall structure or its ability to function as a plastic-binding protein.

Next, we performed an **electrostatic and hydrophobicity assessment** *in silico*. These analyses revealed that BARBIE1 had increased hydrophobicity and a more negative charge compared to BaCBM2. Both characteristics are favorable for enhancing its affinity for hydrophobic plastic surfaces, suggesting that BARBIE1 would be more effective in binding plastics under various environmental conditions.

**Experimental Tests**

We used **size exclusion chromatography with multi-angle light scattering (SEC-MALS)** to analyze the oligomeric states of both BaCBM2 and BARBIE1. The results for BaCBM2 were consistent with the literature, showing predominantly monomeric forms, as well as smaller populations of dimers and tetramers. In contrast, the SEC-MALS results for BARBIE1 revealed a tendency for protein aggregation, with values that did not correspond to dimers, trimers, or tetramers, but rather large protein aggregates. This tendency for aggregation highlighted a potential issue with BARBIE1's stability in solution.

We also used **dynamic light scattering (DLS)**to study BARBIE1’s interaction with plastic particles. The data showed that BARBIE1 formed protein-corona structures around the plastic particles, indicating an increase of the hydrodynamic radius, which could enhance its ability to capture microplastics but also raised concerns about solubility under certain conditions.

**Molecular dynamics simulations** were performed to test BARBIE1’s plastic-binding affinity under different pH levels and temperatures. The simulations demonstrated that BARBIE1 maintained its plastic-binding affinity across a range of environmental conditions, indicating a possible robustness as a plastic-binding protein.

**Finally, circular dichroism (CD)** was used to assess the similarity between BARBIE1 and BaCBM2 as well as its thermal stability. The results indicated that while both proteins exhibited some thermal resistance, BARBIE1 showed reduced ability to refold after unfolding compared to BaCBM2, which retained a higher degree of refolding capacity.

#### Learn

The most significant insight from Cycle 1 came through Integrated Human Practices, where discussions with experts revealed that focusing on water treatment plants (WTPs) might not be the most viable path for implementation. The complexities of incorporating new filtration technologies in WTP infrastructure, combined with regulatory challenges, made us pivot towards household filters. This shift empowers individuals to improve their water quality without waiting for large-scale governmental changes.

From a technical perspective, the SEC-MALS results showed that BARBIE1 had a tendency to aggregate, forming protein clusters instead of remaining in its intended monomeric form. This unexpected behavior raised concerns about the protein’s effectiveness in binding microplastics, as aggregation could hinder its functionality. We realized that to move forward, we needed to address this issue directly.

In response to these findings, we added a new step to our protein design pipeline specifically to prevent protein aggregation. This modification aimed to refine BARBIE1 and ensure that it would remain stable in solution, improving its functionality in real-world applications. Additionally, we began exploring new strategies to prevent BARBIE1 from aggregating, ensuring it would be an efficient and reliable component of our filtration system.

These insights and technical adjustments were critical for guiding the next iteration of the project. By addressing the aggregation issue and refining our approach, we could optimize BARBIE1 for its intended use in household filters, making the system more practical and effective.

### Cycle 2

#### Design

With the valuable insights gained from Cycle 1, we shifted our focus towards developing a filtration system for household use. The initial idea was to create a filter capable of efficiently capturing microplastics. We realized that the filter would need a matrix to hold the plastic-binding proteins (PBPs) in place. Initially, we considered immobilizing our proteins on activated charcoal, which is commonly used in filtration systems. However, to ensure that our strategy could be applied to a wider variety of filter types, we opted for a biological alternative.

Inspired by the iGEM UCopenhagen 2022 team, which used spider silk protein (Nt2RepCt) to create sustainable fishing nets, we decided to explore the use of spidroin for our matrix. Spidroin has a high sustainability footprint and is cost-effective compared to other membranes commonly used for water purification1. Additionally, we found in the literature that nanofibers made from similar materials can be used for water filtration, and spidroin can form hydrogels that allow for the immobilization of proteins2. Furthermore, spider silk has been previously used in water purification for the batch adsorption of copper and nickel, providing a basis for its application in our system³.

For this iteration, we designed a construct combining several key elements: the Nt2RepCt spidroin protein, the SpyTag-SpyCatcher system, and the PBP (either BaCBM2 or BARBIE1). The goal was to create a strong and sustainable matrix that could incorporate our plastic-binding proteins and be integrated into household filters.

* **Nt2RepCt Spidroin Protein:**Chosen for its high sustainability and lower cost, this dual-repeat spider silk protein forms nanofibers that provide the structural backbone of the filtration matrix. The protein’s ability to form hydrogels also allowed us to immobilize the PBPs efficiently within the matrix. Literature also supports the use of spider silk in water purification, particularly for the adsorption of metals like copper and nickel, adding further value to its potential use.
* **PBP (Plastic-Binding Protein):** As in Cycle 1, the PBPs (BaCBM2 or BARBIE1) were at the core of the filtration system, responsible for binding microplastics as water passed through the filter. Their enhanced affinity for plastics, validated in the previous cycle, was expected to perform effectively when attached to the spidroin matrix.
* **SpyTag-SpyCatcher System:** This system was included to covalently link the PBPs to the spidroin matrix. The irreversible bond between SpyTag and SpyCatcher ensured that the plastic-binding proteins would remain securely attached to the matrix, even under varying flow conditions.

This design aimed to create a biologically-based filtration system capable of efficiently capturing microplastics while maintaining the sustainability and cost advantages of spidroin compared to conventional filtration membranes.

#### Build

In the build phase of cycle 2, we made multiple attempts to assemble the **BaCBM2-SpyCatcher** and **BARBIE1-SpyCatcher** constructs using **Golden Gate assembly**. Despite repeated efforts, these assemblies were unsuccessful. During this time, we found new literature that provided a better strategy for ensuring that our proteins would adhere to the spider silk matrix, which led us to abandon the current assembly approach.

For the **Nt2RepCt-SpyTag**, the assembly was successful. The plasmids were transformed via electroporation into *E. coli* DH5α, and the correct assembly was confirmed by Sanger sequencing.

Additionally, we worked on the cloning and transformation of **Barbie1-Cys** and **BaCBM2-Cys**. Both proteins were modified with a cysteine residue to enhance their interaction with the microplastics sensor surface, crucial for amplifying signals in electrochemical measurements. The constructs were assembled using the **Golden Gate method** and transformed into *E. coli* DH5α via electroporation. The correct assembly of both constructs was confirmed by **Sanger sequencing**, but we did not proceed with the purification of **BaCBM2-Cys** in Cycle 2. Instead, we shifted our focus to the next phase, where **BaCBM2-Cys** will be fused to the N-terminal of spidroin for further development in Cycle 3.

#### Test

In this phase, we focused on evaluating the expression and purification of **Nt2RepCt-SpyTag** and **Barbie1-Cys**, as well as analyzing the structural properties of Nt2RepCt using **Circular Dichroism (CD)**, the heating ramp analysis, and **Scanning Electron Microscopy (SEM)**.

**Expression and Purification of Nt2RepCt-SpyTag:**

The expression of **Nt2RepCt-SpyTag** was confirmed in *E. coli* DH5α using SDS-PAGE. However, during the initial purification process using **Immobilized Metal Affinity Chromatography (IMAC)** on a Ni-column, a significant amount of Nt2RepCt appeared in the flow-through, indicating inefficient binding to the column. This suggested the need for optimization in the purification protocol to improve yield and binding efficiency.

**Improved Purification of Nt2RepCt-SpyTag:**

In response to the poor initial purification, we optimized the purification strategy by removing imidazole from **Buffer A** and reducing the imidazole concentration in **Buffer B**. This adjustment, combined with a slower flow rate on the ÄKTA system (0.75 mL/min), greatly improved protein binding to the Ni-column. SDS-PAGE analysis confirmed a higher yield of Nt2RepCt in the elution fractions. A second round of purification was performed to further purify the protein, followed by **Size Exclusion Chromatography (SEC)**, which confirmed the homogeneity of the sample.

**Expression and Purification of Barbie1-Cys:**

Barbie1-Cys expression was also confirmed using SDS-PAGE. However, during the purification process, we encountered challenges regarding the co-elution along with bacterial proteins. Despite using a linear gradient of imidazole for elution, the separation between Barbie1-Cys and *E. coli* proteins was inefficient, hindering further characterization of Barbie1-Cys in this cycle. This result highlighted the need for additional optimization in the purification protocol, particularly to resolve the co-elution issue.

**Circular Dichroism (CD) Analysis:**

Following purification, **CD analysis** was used to examine the secondary structure of **Nt2RepCt** before and after heating. The CD spectrum showed significant changes after the heating ramp, indicating a structural transition, likely associated with the formation of a hydrogel. The heating process altered the secondary structure, consistent with the expected behavior of hydrogel formation, making it a promising candidate for use in water filtration applications.

**Nt2RepCt Heating Ramp:**

To further explore the hydrogel formation properties of Nt2RepCt, we conducted a **heating ramp** analysis, heating the protein from 25°C to 60°C. The results indicated that the conformational changes observed during heating were non-reversible, which is a key characteristic for stable hydrogel formation. This stability at ambient temperature without reverting to the original protein state is crucial for its use in filtration systems.

**Nt2RepCt Hydrogel Scanning Electron Microscope (SEM):**

Finally, the hydrogel formed by Nt2RepCt was analyzed using **Scanning Electron Microscopy (SEM)**. The SEM images revealed an irregular surface topology with potential pore formation, supporting its functionality in filtering applications. This irregularity and roughness of the surface suggest that the protein can create compartments and fibers, which are beneficial for particle filtration.

#### Learn

Cycle 2 provided crucial insights that shaped the direction of our project, particularly in terms of protein expression, purification, and the functional behavior of our constructs.

**1- Nt2RepCt-SpyTag Purification:** The initial purification attempts for **Nt2RepCt-SpyTag** revealed that our initial buffer conditions were suboptimal, leading to significant protein loss in the flow-through. Through trial and error, we learned that modifying the purification buffers, especially removing imidazole from Buffer A and adjusting the imidazole concentration in Buffer B, significantly improved the binding efficiency of Nt2RepCt to the Ni-column. This allowed us to achieve a purer and more concentrated sample of the protein, demonstrating the importance of buffer composition in maximizing protein yield. Additionally, we discovered that the flow rate on the purification system played a crucial role in avoiding premature fiber formation during purification.

**2- Challenges with Barbie1-Cys Purification:**The purification of Barbie1-Cys faced challenges regarding co-elution along bacterial proteins, making it difficult to separate **Barbie1-Cys** from contaminants. Despite various attempts to optimize the purification protocol, we were unable to obtain a sufficiently pure sample for further characterization. This highlighted the need to rethink our approach to producing and purifying Barbie1-Cys. We learned that more stringent purification strategies will be necessary in the next iteration to address the co-elution issues and to ensure we can proceed with functional testing.

**3-Nt2RepCt Hydrogel Formation:** One of the major successes of Cycle 2 was confirming the ability of **Nt2RepCt** to form hydrogels. Using **Circular Dichroism (CD)** and the **heating ramp analysis**, we observed that Nt2RepCt undergoes a non-reversible conformational change when heated, leading to the formation of a stable hydrogel. This discovery is significant for the development of our filtration system, as the formation of a hydrogel matrix provides a solid foundation for immobilizing plastic-binding proteins and ensuring their functionality in water filtration applications. The non-reversible nature of this conformational change suggests that the hydrogel will maintain its structure under real-world conditions, making it an ideal matrix for long-term use in filtration.

**4- Surface Morphology of Nt2RepCt Hydrogel:** The **Scanning Electron Microscopy (SEM)** images provided valuable insights into the surface structure of the hydrogel formed by Nt2RepCt. The irregular surface and pore formation observed in the SEM images are promising features for filtration applications. The creation of a rough, fibrous surface with potential compartments suggests that Nt2RepCt can effectively filter microplastics by trapping particles within its structure. This finding reinforces the potential of Nt2RepCt as a robust matrix for our filtration system.

**Key Takeaways for Next Cycle:**

* **Optimization of Purification Protocols:**e learned that adjusting purification protocols is critical, particularly for difficult-to-purify proteins like **Barbie1-Cys**. More focused efforts on refining the purification strategy will be necessary in Cycle 3.
* **Hydrogel Formation:**The successful hydrogel formation by **Nt2RepCt** offers a solid foundation for immobilizing plastic-binding proteins, making it a crucial element in our filtration system.
* **Continued Exploration of Protein Aggregation:**Further work is needed to prevent protein aggregation, especially for the PBPs, to ensure their functionality when immobilized on the hydrogel matrix.

### Cycle 3

#### Design

Building on the lessons from Cycle 2 and new findings from the literature, we decided to simplify the design of our filtration system. Instead of using the **SpyTag/SpyCatcher** system to attach BaCBM2 and BARBIE1 to the spidroin matrix, we opted for a more straightforward strategy: fusing the **N-terminal of spidroin** directly to these plastic-binding proteins. This decision was driven by evidence that the N-terminal of spidroin is already sufficient to promote hydrogel formation, making the additional attachment mechanism unnecessary.

**Why This Strategy?**

* **Hydrogel Formation:**Literature indicates that the N-terminal domain of spidroin naturally supports hydrogel formation, which is essential for creating a stable filtration matrix. By fusing this domain directly to BaCBM2 and BARBIE1, we expect to maintain the protein's ability to form a hydrogel while also immobilizing the plastic-binding proteins within the matrix. This simplifies the system and reduces the complexity of the assembly.
* **Reducing Aggregation:**One of the major challenges we encountered in Cycle 2 was the aggregation of **BARBIE1**. By fusing BARBIE1 with the N-terminal spidroin, we anticipate that this new configuration will help reduce aggregation, providing a more stable and functional protein within the hydrogel.* **Efficiency and Simplification:**This new design eliminates the need for extra components, such as SpyTag and SpyCatcher, and allows us to produce a single protein that can both form the hydrogel and bind microplastics. This will streamline production and purification, making the overall system more efficient and scalable.

Our expectation with this revised approach is to create a robust, simplified filtration system that reduces the complexity of previous designs and also enhances performance through a more stable and efficient protein-hydrogel integration.

#### Build

In the build phase, we constructed the fusion proteins **Nt-BaCBM2-Cys** and **Nt-Barbie1-Cys** using **Gibson Assembly**, which allowed us to integrate the N-terminal of spidroin directly with the plastic-binding proteins BaCBM2 and BARBIE1, both modified with a cysteine for enhanced interaction with the filtration matrix.

* Nt-BaCBM2-Cys: The biobricks **BBa\_K5396009** (basic) and **BBa\_K5396005** (composite) encode the N-terminal of spidroin (Nt2RepCt) fused to **BaCBM2-Cys**. The construct was generated using Gibson Assembly with the previously constructed plasmids **BBa\_K5396007** and **BBa\_K5396011** as templates. After assembly, the product was transformed into *E. coli* DH5α via electroporation, and successful plasmid construction was confirmed through **Sanger sequencing**.
* Nt-Barbie1-Cys: Similarly, the biobricks **BBa\_K5396010** (basic) and **BBa\_K5396006** (composite) encode the N-terminal of spidroin fused to **Barbie1-Cys**. This construct was also generated using Gibson Assembly with the plasmids **BBa\_K5396008** and **BBa\_K5396011** as templates. The assembled product was transformed into *E. coli* DH5α via electroporation, and the successful construction was confirmed by **Sanger sequencing**.

#### Test

During Cycle 3, several tests were performed to evaluate the expression, purification, and structural characterization of **Nt-BaCBM2-Cys** and **Nt-Barbie1-Cys:**

**Purification of Nt-BaCBM2-Cys:**

The first attempt to purify **Nt-BaCBM2-Cys** using **Immobilized Metal Affinity Chromatography (IMAC)** on a Ni-column resulted in co-elution with *E. coli* proteins. A single peak was observed on the chromatogram, and SDS-PAGE confirmed the presence of both the target protein and bacterial contaminants.

**Refinement of Purification:**

In the second purification attempt, the flow rate was reduced to 0.75 mL/min, leading to improved peak separation. **SDS-PAGE** analysis of the eluted fractions confirmed higher concentrations of purified **Nt-BaCBM2-Cys** in peaks 3 and 4, with fewer contaminants.

**Expression Tests:**

To optimize the expression of **Nt-BaCBM2-Cys** and **Nt-Barbie1-Cys**, small-scale induction tests were conducted. Samples were taken at 0h, 3h, 4h, and 5h after IPTG induction. SDS-PAGE results showed a slight increase in protein expression in the samples collected after 3-5 hours.

**Circular Dichroism (CD) Analysis:**

With the newly purified **Nt-BaCBM2-Cys**, CD analysis was performed to characterize its secondary structure. The spectrum showed a notable similarity between **Nt-BaCBM2-Cys** and **Nt2RepCt-SpyTag**, particularly in the range of 210 to 250 nanometers, indicating a structural contribution from both domains. In contrast, **BaCBM2-RFP-3xMad10** displayed significant differences in the CD spectrum, particularly at lower wavelengths. Between 205 and 210 nm, **Nt-BaCBM2-Cys** showed lower absorbance compared to both **Nt2RepCt** and **BaCBM2**, suggesting that the fusion of the BaCBM2-Cys and Nt domains affects the overall secondary structure.

#### Learn

Cycle 3 provided valuable insights into the challenges of purifying and expressing both Nt-BaCBM2-Cys and Nt-Barbie1-Cys, as well as offering a deeper understanding of their structural properties through Circular Dichroism (CD) analysis.

* **Purification Challenges:** The initial difficulty in separating Nt-BaCBM2-Cys from *E. coli* contaminants highlighted the need for optimization of the purification process. By adjusting the flow rate during IMAC purification, we improved peak separation, but overall protein yield remained low. This indicates that further refinements are necessary, possibly involving modifications to the buffer composition or the use of different purification strategies.
* **Expression Optimization:** Expression tests conducted for both Nt-BaCBM2-Cys and Nt-Barbie1-Cys showed a slight increase in protein production after 3-5 hours of IPTG induction. However, the constitutive activity of the AB\_T7\_lacO promoter suggests that it may not be fully responsive to IPTG, limiting the increase in expression levels for both proteins. This insight suggests that alternative expression systems or promoters may be needed to achieve higher yields in future cycles.
* **Structural Insights from Circular Dichroism (CD):** The CD analysis of Nt-BaCBM2-Cys provided key insights into its secondary structure. The similarities observed between Nt-BaCBM2-Cys and Nt2RepCt-SpyTag suggest that the N-terminal spidroin domain contributes significantly to the protein’s overall structure. However, the contrast between Nt-BaCBM2-Cys and BaCBM2-RFP-3xMad10, especially at lower wavelengths, indicated that the fusion of BaCBM2-Cys with the N-terminal of spidroin altered the structural profile of the protein. This finding is crucial for understanding how the fusion impacts the behavior of the protein in the hydrogel matrix and its binding capacity to microplastics.
* **Aggregation Comparison Between Cycles 1 and 3:** Upon comparing the aggregation results from Cycle 1 and Cycle 3, we observed that the aggregation score for BARBIE1 did not improve significantly between these cycles. This indicated that despite the design modifications, the fusion did not resolve the aggregation issue as much as expected. To address this, the next step is to add an MBP (Maltose-Binding Protein) tag, which has been validated *in silico* to improve solubility and could potentially reduce aggregation in future experiments.
* **Hydrogel Formation:** Although some purified Nt-BaCBM2-Cys was obtained, the yield was still insufficient to perform hydrogel formation tests. This reinforced the importance of optimizing the expression and purification steps to generate enough protein for functional characterization and hydrogel testing in the future.

**Next Steps:**

* **Further Refinement of Purification:** Additional experiments will focus on fine-tuning the purification protocol to achieve higher yields and better separation of the target protein from contaminants.
* **Promoter Investigation:** We will explore alternative promoters or expression systems to improve the responsiveness to IPTG and boost protein production for both Nt-BaCBM2-Cys and Nt-Barbie1-Cys.
* **MBP Tag Addition:** To improve solubility and reduce protein aggregation, we plan to incorporate an MBP (Maltose-Binding Protein) tag in future iterations, which has already shown promising results in *in silico* analysis.
* **Pipeline for Multiple Pollutants:** The success of our protein engineering pipeline has inspired the development of a new approach for the B.A.R.B.I.E project: a toolbox for customizable protein design. This pipeline, capable of designing proteins with enhanced affinity for specific pollutants, offers a versatile solution not only for iGEM teams but also for addressing real-world environmental challenges. It can be adapted and tested for filtering a wide range of pollutants from water, extending beyond microplastics.
* **Gene Library for High-Throughput Testing:** As a result of our novel protein design pipeline, we have synthesized a gene library containing over 10,000 different proteins suitable for expression in Saccharomyces cerevisiae. Over the next few months, we will perform high-throughput screening to evaluate the efficiency of these proteins in binding and removing different types of pollutants, marking a significant step forward in the development of this customizable filtration system.

## Detection Module

Just like our bioremediation proposal, our sensing proposal has also undergone several changes since last year. In iGEM Design League, our sensing proposal consisted only of an electrochemical impedance sensor (EIS). Since the focus was on water treatment plants, the goal was to sense microplastics approximately 1 mm in diameter using a microfluidic system. This sensor would be placed upstream and downstream relative to the bioremediation proposal, aiming to estimate the amount of microplastics present in the water before and after the treatment process. Although promising, the suggested sensing proposal still had certain limitations regarding the potential manufacturing cost of each sensor and the detection of plastic particles smaller than 200 μm.

This year, the B.A.R.B.I.E project has shifted its focus toward detecting micro- and nanoplastics that pass through conventional water treatment processes and even household filters. In this context, the average particle size becomes approximately 1 μm. Moreover, since the proposal is focused toward household applications, the cost of the device becomes a crucial factor.

### Cycle 1

#### Design

Cycle 1 consisted of a sensing proposal based solely on the electrochemical response of micro- and nanoplastics. The design stages were divided as follows:

* **Sensor architecture:** A three-electrode architecture was chosen: working, counter, and reference electrodes. This configuration allows for various electrochemical measurements to be performed quickly and reproducibly.
* **Detection method:** The initial idea was to explore impedance spectroscopy measurements in a solution of Na₂SO₄. Later, the focus shifted to exploring square-wave voltammetry measurements in a solution of K₂NO₃ with K₃[Fe(CN)₆]/K₄[Fe(CN)₆].

#### Build

Therefore, based on the established design, the electrochemical devices were microfabricated in a cleanroom environment at the Brazilian Nanotechnology National Laboratory (LNNano) through the following fabrication steps:

* Glass substrate preparation
* Hexamethyldisilazane (HMDS) adhesion layer
* Photoresist deposition
* Photolithography and development
* Metal layer deposition
* Liftoff process
* Surface cleaning and hydrophilization
* SU-8 photoresist deposition
* Photolithography for SU-8
* Development of SU-8
* Final cleaning

Additionally, the experimental setup for the measurements was assembled using the portable PalmSens4 potentiostat, connectors, and a notebook for data storage and monitoring. Tests with different concentrations of polystyrene (PS) particles were prepared to evaluate the sensor’s performance.

#### Test

Therefore, the following tests were conducted:

* Electrochemical Impedance Spectroscopy (EIS) measurements for Na₂SO₄ and SWV for K₃[Fe(CN)₆]/K₄[Fe(CN)₆]: It was possible to evaluate the behavior of the solution using impedance spectroscopy.
* Analytical curve with EIS and Na₂SO₄, and with Square Wave Voltammetry SWV and K₃[Fe(CN)₆]/K₄[Fe(CN)₆]: The linearity in detecting PS particles was assessed.
* Scanning Electron Microscopy (SEM): The microfabricated device was thoroughly examined to understand the precision of the fabrication process.

#### Learn

From the results obtained in the tests, it was noted that there was some difficulty in finding a correlation between the impedance measurements and the PS concentration. Based on this, our team learned that square wave voltammetry (SWV) might be more advantageous for quantifying microplastics in our system due to its precision, simplicity, and speed. Moreover, for the students involved in the practical work, this cycle was extremely important as it provided a first experimental experience with several electrical measurements, which facilitated the development of subsequent cycles.

### Cycle 2

#### Design

After the first cycle, it was observed that the electrochemical sensor without biological modification showed limitations in the precise quantification of microplastics (MNPs), especially at lower concentrations. Thus, in the second cycle, the inclusion of a biological element was proposed to increase the sensor’s selectivity: the BaCBM2 protein, known for its high affinity for plastics. The objective was to test two main approaches for protein immobilization on the working electrode surface:

* **Dropcasting:** A simple method of protein adsorption, where a drop of the BaCBM2 solution is deposited directly on the electrode, allowing fixation through physical interaction.
* **Self-Assembled Monolayers (SAMs):** Structured modifications on the electrode surface using L-cysteine, cysteamine, and graphene oxide, with the goal of promoting a stronger and more stable protein attachment, thereby amplifying the electrochemical response.

Based on the protein-plastic binding characteristics, the use of the Langmuir model was planned to determine the dissociation constant (Kd), which allows quantification of the affinity between the proteins and plastic particles.

#### Build

The construction phase involved the preparation and modification of the sensors using the two immobilization methods:

* **Drop casting:** The BaCBM2 protein was adsorbed directly onto the electrode surface by depositing the protein solution followed by an incubation period. The sensor was then washed and subjected to electrochemical measurements using square wave voltammetry (SWV).
* **Self-Assembled Monolayers (SAMs):** The electrode surface was modified using three different agents (L-cysteine, cysteamine, and graphene oxide) to form self-assembled monolayers that facilitate the immobilization of BaCBM2. Each agent was incubated with the sensor for 15 hours at 4°C, followed by protein deposition, activation and the application of SuperBlock, to block non-specific binding sites.

Additionally, assays with progressively increasing concentrations of polystyrene nanobeads (PSNBs) (0.01 to 100 mg/L) were prepared to create analytical curves and evaluate the sensor’s performance.

#### Test

The modified sensors underwent a series of tests:

* SWV after modification: Square wave voltammetry (SWV) measurements were taken before and after the immobilization of the BaCBM2 protein. A noticeable decrease in current was observed after protein adsorption, indicating that the protein successfully adhered to the electrode surface.
* XPS (X-ray Photoelectron Spectroscopy): Surface characterization was performed to confirm the presence and homogeneous distribution of the proteins and SAMs on the electrode.
* Analytical curve: Both the drop casting method and the SAMs approach generated analytical curves based on microplastic concentrations. The SAM modified with cysteamine resulted in a curve with a coefficient of determination (R²) of 0.99, showing excellent reproducibility and low variations between measurements.
* Determination of Kd: The dissociation constant (Kd) was obtained using the Langmuir model to quantify the affinity between the BaCBM2 protein and polystyrene particles.

#### Learn

The results demonstrated a significant improvement in the quantification of microplastics compared to the previous cycle. The SAM modification, particularly using cysteamine, proved to be the most efficient, resulting in a more precise and reproducible electrochemical response, with an R² of 0.99. The use of immobilized proteins allowed for more accurate quantification of microplastics and opened the possibility of determining the dissociation constant (Kd), which could be valuable for more detailed studies of protein-particle affinity. Furthermore, this cycle highlighted the importance of the protein-plastic interaction in the sensor efficiency, suggesting that future adjustments to immobilization conditions and sensor design could further enhance performance.

### Cycle 3

#### Design

Cycle 3 explored a new approach for the sensor, utilizing the process of protein corona formation on micro and nano plastic particles (MNPs). The main concept involved the interaction of MNPs with the BaCBM2 protein in solution before deposition onto the working electrode. This bulk interaction process allowed for the formation of protein layers around the MNPs, creating what is known as a protein corona.

The choice of this methodology was motivated by the potential to enhance the selectivity and detection of MNPs with pre-adsorbed proteins, which can help capture the target particles more effectively. Additionally, the expression and purification of the BaCBM2 protein with a cysteine coating were considered to improve interaction with the surface of the MNPs.

#### Build

To construct this biosensor, the following steps were carried out:

* **Solution Preparation:** MNPs and the BaCBM2 protein were mixed in PBS solution and agitated for 10 minutes to ensure the formation of protein coronas around the particles.
* **Dynamic Light Scattering (DLS) Measurements:** DLS was conducted to evaluate the particle size and confirm the formation of the corona around the MNPs. These measurements allowed for the identification of changes in hydrodynamic diameter, suggesting the involvement of the protein.
* **Computational and Analytical Modeling:** A theoretical model was developed to predict the formation of the coronas and the interaction between the MNPs and the proteins in solution, based on the experimental conditions.
* **Electrochemical Measurements:** After the formation of the coronas, part of this solution was deposited directly onto the sensors, followed by the addition of an electrolyte to establish contact between the electrodes. Square wave voltammetry (SWV) measurements were then performed.

#### Test

The tests conducted yielded the following results:

* **DLS Results:** The DLS measurements confirmed the formation of protein coronas around the MNPs, with a significant increase in hydrodynamic diameter, demonstrating that the adsorption process was effective.
* **Computational Analysis:** The theoretical model developed to describe the formation of the coronas was consistent with the results observed in the DLS measurements, indicating that the formation of the protein layers can be reliably predicted.
* **SWV Measurements:** The analytical curves obtained from the SWV measurements revealed a decrease in the coefficient of determination (R² = 0.40), indicating a weak correlation between the concentration of MNPs and the electrochemical response. Additionally, the standard deviations in the measurements increased, negatively impacting the reproducibility of the results.

#### Learn

Despite the unsatisfactory results for the quantification of MNPs, Cycle 3 provided valuable insights into the formation of protein coronas. The technique was confirmed by DLS, demonstrating effective interaction between the proteins and the MNPs, which can be beneficial for future sensor optimizations.

Although the initial strategy of expressing and purifying the BaCBM2 protein with a cysteine solution was not tested, there is significant potential to integrate this approach in upcoming cycles to ensure greater accuracy in the corona deposition process.

The primary limitation identified was the lack of precision and reproducibility in quantifying MNPs through electrochemical measurements. This suggests that adjustments in the interaction time between the proteins and the MNPs, as well as the incubation time on the sensor surface, could enhance sensor performance in future cycles. In summary, this cycle paved the way for new investigations and optimizations, particularly concerning the behavior of protein coronas and their influence on electrochemical outcomes.

### References

1 Peydayesh, M. & Mezzenga, R. Protein nanofibrils for next generation sustainable water purification. Nat. Commun. 12, 3248 (2021). <https://doi.org/10.1038/s41467-021-23388-2>
2 Arndt, T., Jaudzems, K., Shilkova, O. et al. Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform. Nat. Commun. 13, 4695 (2022). <https://doi.org/10.1038/s41467-022-32093-7>
3 Pelit, L., Ertaş, F. N., Eroğlu, A. E., Shahwan, T. & Tural, H. Biosorption of Cu(II) and Pb(II) ions from aqueous solution by natural spider silk. Bioresour. Technol. 102, 8807–8813 (2011). <https://doi.org/10.1016/j.biortech.2011.07.013>
4 Colson, B. C. & Michel, A. P. M. Flow-through quantification of microplastics using impedance spectroscopy. ACS Sens. 6, 1 (2021). <https://doi.org/10.1021/acssensors.0c02223>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block cover\_image %}
![Experiments Cover](https://static.igem.wiki/teams/5396/capas/experiments.webp)
{% endblock %}
{% block page\_content %}

Welcome to the Experiments section of the BARBIE Project. Here, you will find comprehensive protocols and procedures detailing the experiments conducted throughout this year’s journey. Each section is crafted to guide you through the methodologies and techniques we employed, featuring an interactive protocol\* for deeper exploration and learning of protein production.

## LB (Luria Broth) Agar Plates

#### Materials

* Equipment: Petri Dishes, Erlenmeyer Flask, Autoclave, Sterile Pipettes
* LB Broth (To prepare 1 L ⇒ 10 g Tryptone; 5 g Yeast Extract; 10 g NaCl; 20 g Bacto Agar and complete with water)
* Antibiotic (if needed)

#### Steps

1. **Prepare LB Broth:** Combine 10 g tryptone, 5 g yeast extract, 10 g NaCl, and 20 g agar in 800 mL sterile water. Adjust volume to 1 L and pH to 7.0-7.5.
2. **Sterilisation:** Autoclave the broth at 121°C for 25 minutes.
3. **Pour Plates:** Add the antibiotics if needed. Pour ~20 mL of agar mixture into sterile Petri dishes and allow to solidify.
4. **Storage:** Invert plates to prevent condensation, wrap or bag them, label, and store at 4°C.

## Electrocompetent Cells

#### Materials

* Bacterial Strain: E. coli (e.g., DH5α)
* Growth Medium: Luria Broth (LB)
* Glycerol: 10% sterile solution
* Equipment: Erlenmeyer Flasks, Centrifuge, Ice or Cold Room (4°C), Sterile Microcentrifuge Tubes, Electroporation Cuvettes

#### Steps

1. **Inoculate Bacteria:** Streak a single colony of E. coli on an LB agar plate and incubate overnight at 37°C.
2. **Inoculate a Single Colony:** Transfer into 3-5 mL of LB broth and shake overnight at 37°C.
3. **Dilute and Grow:** Dilute the overnight culture 1:100 to 1:500 in fresh LB broth (0.5 to 1 L) and grow until OD600 reaches ~0.4-0.6 (mid-log phase), approximately 3-6 hours.
4. **Harvest Cells:** Cool the culture on ice for 10-15 minutes.
5. **Transfer and Centrifuge:** Transfer to pre-chilled centrifuge bottles and centrifuge at 5000 rpm for 5-10 minutes at 4°C to pellet cells.
6. **Wash Cells:** Resuspend pellet in equal volume of ice-cold sterile deionized water and centrifuge again.
7. **Repeat Wash:** Repeat the wash step two more times, gradually reducing the volume of water to about half the original culture volume.
8. **Glycerol Wash:** Resuspend the final pellet in ~20 mL ice-cold sterile 10% glycerol solution.
9. **Centrifuge Again:** Centrifuge at 10,000 rpm for 10-15 minutes at 4°C.
10. **Final Resuspension:** Resuspend the pellet in a small volume of ice-cold sterile 10% glycerol (3-4 mL per liter of original culture).
11. **Aliquot:** Aliquot suspension into pre-chilled microcentrifuge tubes (e.g., 40 µL per tube).
12. **Storage:** Freeze aliquots in liquid nitrogen or dry ice/ethanol bath for long-term storage. Store at -80°C until needed.
13. **Using Competent Cells:** Thaw an aliquot on ice, add salt-free DNA (1-5 µL), mix gently, and transfer to an electroporation cuvette. Electroporate as per manufacturer's instructions, then immediately add recovery medium (e.g., SOC) and incubate at 37°C for ~1 hour before plating on selective media.

## Chemocompetent Protocol

#### Materials

* Bacterial Strain: E. coli (e.g., DH5α)
* Growth Medium: Luria Broth (LB)
* Glycerol: 15% sterile solution
* Calcium Chloride (CaCl₂): 100 mM solution, ice-cold
* Equipment: Erlenmeyer Flasks, Centrifuge, Ice or Cold Room (4°C), Sterile Microcentrifuge Tubes, Spectrophotometer for Measuring Optical Density

#### Steps

1. **Inoculate Bacteria:** Start with a single colony of E. coli grown on an LB agar plate. Inoculate into 10 mL of sterile LB broth and incubate overnight at 37°C with shaking (200 rpm).
2. **Dilute and Grow:** Dilute the overnight culture by transferring 1 mL into 99 mL fresh sterile LB broth. Incubate at 37°C with shaking until OD600 reaches ~0.3-0.5 (mid-log phase), approximately 3-6 hours.
3. **Harvest Cells:** Cool the culture on ice for ~10-15 minutes.
4. **Transfer and Centrifuge:** Transfer to sterile centrifuge tubes and centrifuge at ~7000 rpm for 10 minutes at 4°C to pellet cells. Discard supernatant carefully.
5. **Wash Cells:** Resuspend pellet in 20 mL ice-cold sterile 100 mM CaCl₂ solution. Gently mix and chill on ice for another 15 minutes.
6. **Centrifuge Again:** Centrifuge at the same speed and temperature, then discard supernatant.
7. **Resuspend Pellet:** Resuspend pellet in 5 mL ice-cold sterile 100 mM CaCl₂ supplemented with 15% glycerol and gently mix.
8. **Aliquot Cells:** Divide resuspended cells into small aliquots (e.g., 50 µL) in sterile, ice-cold microcentrifuge tubes.
9. **Storage:** Store aliquots at -80°C for long-term storage. Cells remain competent for at least one year.
10. **Transformation Efficiency Testing (Optional):** Transform aliquots with known amount of plasmid DNA containing a positive selection marker and count transformant colonies after plating on selective media.

## Golden Gate Cloning

#### Materials

* DNA Fragments:
  + Plasmid Backbone (Destination Vector)
  + Insert Fragments (PCR Products or Pre-cloned)
* Enzymes:
  + Type IIS Restriction Enzyme (e.g., BsaI or BsmBI)
  + T4 DNA Ligase
* Buffers:
  + 10X T4 DNA Ligase Buffer
  + Water: Nuclease-Free Water
* Equipment:
  + Thermocycler
  + Ice or Cold Room
  + Microcentrifuge Tubes

#### Steps

1. **Design the Assembly:** Ensure each insert and plasmid backbone have compatible overhangs generated by the chosen Type IIS enzyme. Recognition sites should flank the desired insert region.
2. **Prepare the Reaction Mix:** In a total volume of 10 µL, combine:
   * Plasmid Backbone (75 ng/µL) ⇒ 1 µL
   * Insert Fragments (75 ng each) ⇒ Variable
   * 10X T4 DNA Ligase Buffer ⇒ 1 µL
   * T4 DNA Ligase (400 U/µL) ⇒ 1.25 µL
   * Type IIS Restriction Enzyme ⇒ 0.5 µL
   * Nuclease-Free Water ⇒ To 10 µL
3. **Set Up the Assembly Reaction:** Combine all components in a microcentrifuge tube. Mix and briefly centrifuge to collect the liquid at the bottom.
4. **Perform the Assembly Reaction:** Use thermal cycling conditions based on the number of inserts:
   * For 1 Insert ⇒ 37°C for 5 min (cloning) OR 37°C for 1 h AND 60°C for 5 min
   * For 2-4 Inserts ⇒ 37°C for 1 hr AND 60°C for 5 min
   * For 5-10 Inserts ⇒ (37°C for 1 min AND 16°C for 1 min) x30 AND 60°C for 5 min
   * For >10 Inserts ⇒ (37°C for 5 min AND 16°C for 5 min) x30 AND 60°C for 5 min
5. **Transform Competent Cells:** After assembly, transform the mixture into competent E. coli cells using heat shock or electroporation. Plate-transformed cells on selective media to identify successful clones.

**Notes:**

* Ensure total volume of unpurified PCR fragments in assembly reaction does not exceed 20% of total volume.
* Use equimolar concentrations of all fragments for optimal results.
* Include a positive control to verify assembly efficiency.

## Gibson Cloning

#### Materials

* DNA Fragments:
  + Linearized Vector (Plasmid Backbone)
  + Insert Fragments (PCR Products or Other Linear DNA)
* Gibson Assembly Master Mix
* Buffers and Water:
  + Nuclease-Free Water
* Equipment:
  + Thermocycler
  + Ice or Cold Room
  + Microcentrifuge Tubes

#### Steps

1. **Design Primers for PCR Amplification:** Ensure overlap sequences (20-40 bp) for assembly of adjacent fragments.
2. **Prepare the Gibson Assembly Reaction:** In a 20 µL volume, combine:
   * Gibson Assembly Master Mix ⇒ 10 µL
   * DNA Fragments (0.02–0.5 pmols for 1-2 fragments, or 0.2–1.0 pmols for 4-6 fragments) ⇒ Variable
   * Nuclease-Free Water ⇒ To 20 µL
3. **Incubate the Reaction:** Incubate at 50°C for:
   * 15 minutes if assembling 2-3 fragments.
   * 60 minutes if assembling 4-6 fragments.
4. **Transform Competent Cells:** After incubation, place reaction on ice. Transform into competent cells using standard protocols. Plate-transformed cells on selective media to identify successful clones.

**Notes:**

* Total volume of unpurified PCR fragments should not exceed 20% of the total reaction volume.
* Use equimolar concentrations of all fragments for optimal results.
* Include a positive control to verify assembly efficiency.

## Transformation by Electroporation

#### Materials

* Electrocompetent Cells: E. coli
* DNA Sample: Plasmid or Other DNA for Transformation
* Electroporation Cuvettes: 1 mm Gap
* Recovery Medium
* Buffers and Water:
  + Sterile Water for Dilutions
* Equipment:
  + Electroporator
  + Ice or Cold Room
  + Microcentrifuge Tubes
  + 17 mm x 100 mm Round-Bottom Culture Tubes

#### Steps

1. **Preparation:**
   * Pre-warm selective agar plates at 37°C for at least 1 hour.
   * Place recovery medium in a 37°C water bath.
   * Chill electroporation cuvettes and microcentrifuge tubes on ice.
2. **Thaw Competent Cells:** Thaw electrocompetent E. coli cells on ice for ~10 minutes. Gently mix by flicking the tube.
3. **Prepare DNA Solution:** Dilute a control plasmid (e.g., pUC19) to a final concentration of 10 pg/µL using sterile water.
4. **Mix Cells and DNA:** Transfer 25 µL competent cells to a chilled microcentrifuge tube. Add 1 µL DNA solution and gently mix without introducing bubbles.
5. **Transfer to Cuvette:** Carefully transfer cell/DNA mixture into chilled electroporation cuvette, ensuring cells settle without bubbles.
6. **Electroporate:** Use the following conditions:
   * Voltage: 2.0 kV
   * Resistance: 200 Ω
   * Capacitance: 25 µF
7. **Recovery:** Immediately add 975 µL pre-warmed recovery medium to cuvette and gently mix by pipetting up and down twice.
8. **Transfer and Incubate:** Transfer mixture to pre-warmed round-bottom culture tube. Incubate at 37°C with shaking (250 rpm) for ~1 hour.
9. **Plating:** Dilute cells as necessary and spread 100-200 µL onto pre-warmed selective agar plates. Incubate overnight at 37°C.

## Transformation by Heat-shock

#### Materials

* Competent Cells: E. coli (e.g., DH5α or Other Competent Strains)
* DNA Sample: Plasmid DNA (1 pg to 100 ng)
* Recovery Medium: LB Broth or SOC
* Selection Plates: LB Agar Plates with Appropriate Antibiotic
* Equipment:
  + Ice or Cold Room
  + Water Bath or Heat Block set at 42°C
  + Incubator set at 37°C
  + Microcentrifuge Tubes
  + Pipettes and Sterile Tips

#### Steps

1. **Preparation:**
   * Thaw competent E. coli cells on ice for ~10 minutes.
   * Pre-warm LB broth or SOC medium in a water bath to 37°C.
2. **Mix Cells and DNA:** Add 1-5 µL plasmid DNA to thawed competent cells (typically 50 µL) in a microcentrifuge tube. Gently flick to mix; do not vortex.
3. **Incubate on Ice:** Incubate mixture on ice for 30 minutes to allow DNA uptake.
4. **Heat Shock:** Transfer tube to a water bath or heat block set at 42°C for exactly 30-90 seconds (30 seconds typical).
5. **Cool on Ice:** Immediately return tube to ice and cool for an additional 2-5 minutes.
6. **Add Recovery Medium:** Add 500 µL pre-warmed LB broth or SOC medium to cells.
7. **Incubate:** Incubate at 37°C with shaking (180 rpm) for 1 hour to allow recovery and expression of antibiotic resistance.
8. **Plating:** After incubation, gently mix cells and perform serial dilutions if necessary. Spread 50-100 µL of cell suspension onto pre-warmed selective agar plates. Incubate overnight at 37°C.

**Notes:**

* Ensure all materials are sterile to prevent contamination.
* Use freshly prepared competent cells and avoid prolonged heat exposure during heat shock to maintain transformation efficiency.
* Adjust DNA and competent cell volumes based on experimental needs; larger volumes may require longer incubation times.

## Agarose Gel Electrophoresis

#### Materials

* Agarose Powder (1% or 0.8% depending on fragment size)
* Buffer Solution: TAE or TBE
* DNA Samples (PCR Products, etc.)
* Loading Buffer
* Ethidium Bromide or GelRed (optional for visualization)
* Gel Casting Tray and Comb
* Electrophoresis Apparatus
* Power Supply
* UV Transilluminator (for visualization)

#### Steps

1. **Prepare the Agarose Gel:** Weigh agarose (1 g for 100 mL at 1%). Mix with TAE/TBE buffer and dissolve by microwaving. Cool to ~55°C, pour into casting tray, and let solidify.
2. **Prepare DNA Samples:** Mix DNA with loading buffer. Include a molecular weight ladder as a control.
3. **Load the Gel:** Place solidified gel in electrophoresis tank and cover with running buffer. Load DNA samples and ladder into wells.
4. **Run the Gel:** Connect to power supply and run at 80-150 V until loading dye migrates 75-80% down the gel.
5. **Visualize DNA:** Stain with ethidium bromide if necessary. Use UV transilluminator to visualize and document DNA bands.

## Sanger Sequencing

#### Materials

* Template DNA: Plasmid or PCR Product
* Primers: Specific to Target Region
* BigDye Terminator v3.1 Cycle Sequencing Kit
* Nuclease-Free Water
* Capillary Electrophoresis System
* Microcentrifuge Tubes

#### Steps

1. **Prepare DNA Template:** Purify and measure DNA concentration (aim for 10-100 ng/µL).
2. **Set Up Sequencing Reaction:** Combine in microcentrifuge tube: Template DNA (10-100 ng), primers, BigDye Terminator mix, and nuclease-free water as per kit instructions.
3. **Perform Sequencing Reaction:** Use thermal cycling conditions specified in the BigDye Terminator kit, typically including denaturation, annealing, and extension phases.
4. **Clean Up Reaction:** Purify to remove unincorporated dNTPs and enzymes.
5. **Prepare for Capillary Electrophoresis:** Resuspend purified product in loading buffer, heat at 95°C for 5 min, then place on ice.
6. **Run Capillary Electrophoresis:** Load samples into the capillary system and run according to the manufacturer's instructions.
7. **Analyze Results:** Use software to analyze chromatograms and determine the nucleotide sequence.

## Restriction Digestion

#### Materials

* DNA Sample: Plasmid or PCR Product
* Restriction Enzyme: Specific to Recognition Sites in DNA
* Buffer: Appropriate Buffer for Restriction Enzyme (usually supplied with enzyme)
* BSA (Bovine Serum Albumin): If required by enzyme
* Nuclease-Free Water
* Microcentrifuge Tubes
* Pipettes and Tips

#### Steps

1. **Prepare Reaction Mix:** In a microcentrifuge tube, combine:
   * Nuclease-Free Water ⇒ To reach final volume (e.g., 16-16.5 µL for a 20 µL reaction)
   * 10X Restriction Buffer ⇒ 2 µL
   * DNA Sample ⇒ 1 µL (~1 µg)
   * Restriction Enzyme ⇒ 0.5-1 µL (5-10 U)
2. **Mix Components:** Gently mix by pipetting up and down. Avoid bubbles. Briefly centrifuge the tube to collect all liquid at the bottom.
3. **Incubate Reaction:** Incubate at the optimal temperature for the specific restriction enzyme (usually 37°C) for 1 to 16 hours, depending on enzyme activity and DNA complexity.
4. **Stop Reaction (if necessary):** Some enzymes can be heat-inactivated. If required, incubate at 65°C for 20 minutes or according to the enzyme's specifications.
5. **Analyze Digested DNA:** Run an aliquot of the digested sample on an agarose gel to confirm successful digestion.
6. **Purify Digested DNA (if needed):** Use a purification kit or perform ethanol precipitation to purify the digested DNA for subsequent applications.

## Western Blot

#### Materials

* Filter Paper
* Electroblotting Equipment
* Cassettes
* PBS
* Samples (Protein SDS-PAGE Gel)
* Blocking Solution: 5% Skim Nonfat Dry Milk Powder in PBS-T
* Primary Antibody Solution: 1:1000 dilution of primary antibody (mouse anti-hexahis antibody) in blocking solution
* Secondary Antibody Solution: 1:5,000 dilution of secondary antibody (goat anti-mouse HRP conjugated) in blocking solution

#### Steps

1. **Make a Sandwich:** Layer filter papers, place the membrane on top of the filter paper, transfer the SDS-PAGE gel onto the membrane, and cover with additional filter papers.
2. **Transfer the Sandwich to the Blotting Machine:** Place the assembled sandwich into the "Trans-Blot Turbo" machine following the manufacturer's instructions. Select the appropriate settings based on the membrane type (mini or maxi).
3. **Transfer Proteins:** Run the blotting process as per the machine's guidelines. Once completed, carefully remove the blotted membrane.
4. **Block the Membrane:** Incubate the membrane in the blocking solution (5% skim nonfat dry milk powder in PBS-T) for 1 hour at room temperature or overnight at 4°C with shaking to prevent non-specific binding.
5. **Primary Antibody Incubation:** Incubate the membrane with the primary antibody solution (1:1000 dilution of mouse anti-hexahis antibody in blocking solution) for 1 hour at room temperature or overnight at 4°C with shaking.
6. **Wash the Membrane:** Wash the membrane three times for 5 minutes each with PBS-T to remove unbound primary antibodies.
7. **Secondary Antibody Incubation:** Incubate the membrane with the secondary antibody solution (1:5,000 dilution of goat anti-mouse HRP conjugated secondary antibody in blocking solution) for 1 hour with shaking.
8. **Wash the Membrane Again:** Wash the membrane three times for 10 minutes each with PBS-T to remove unbound secondary antibodies.
9. **Add Color-Developing Solution:** Apply the color-developing solution to the blot to visualize the protein bands.
10. **Incubate in the Dark:** Incubate the blot with the color-developing solution in the dark for no more than 5 minutes to prevent overdevelopment.
11. **Visualize the Blot:** Place the blot on the gel imaging system to visualize and document the protein bands.

## BCA’s Assay

#### Materials

* BCA Protein Assay Kit and Samples
* 96-well Microplate
* Protein Standards

#### Steps

1. **Prepare Protein Standards:** Create a series of protein standards with different BSA concentrations as per the kit instructions.
2. **Add Standards and Samples:** Add 10 µL of each standard and your GFP elution sample “E” into designated wells of the microplate in duplicate.
3. **Prepare Working Solution:** Mix Reagent A and Reagent B in a 50:1 ratio to create the working solution.
4. **Add Working Solution:** Add 200 µL of the working solution to each well containing standards and samples. Mix gently to ensure thorough mixing.
5. **Incubate:** Place the microplate in a 37ºC incubator for 30 minutes to allow color development.
6. **Measure Absorbance:** After incubation, measure the absorbance of each well at 562 nm using a plate reader.
7. **Calculate Protein Concentration:** Generate a standard curve using the protein standards and apply the equation to determine the protein concentration of your samples.

## SDS-PAGE Gel

#### Materials

* 1X Running Buffer (0.5 mM Tris base, 192 mM glycine, 0.1% SDS, pH 8.3)
* Sample Buffer (3X) - Blue
* Ladder (usually 1 Kb)
* Resolution (Bottom Gel):
  + Water
  + 1.5 M Tris-HCl pH 8.8
  + 30% Acrylamide
  + 10% SDS
  + 10% Ammonium Persulfate (APS)
  + TEMED
* Stacking (Top Gel):
  + Water
  + 0.5 M Tris-HCl pH 6.8
  + 30% Acrylamide
  + 10% SDS
  + 10% Ammonium Persulfate (APS)
  + TEMED
* Cassettes (Glass and Plastic)
* Alcohol 98%

#### Steps

1. **Prepare the Gel:** Calculate the required amount of polyacrylamide (8-15%) based on your needs. Fill the cassettes with the resolution (bottom) gel mix and add a few drops of 98% alcohol to keep the gel straight. Allow the gel to polymerize for 30 to 60 minutes.
2. **Prepare the Stacking Gel:** Remove the alcohol and pipette the stacking (top) gel mix into the well area. Allow the stacking gel to polymerize for at least 45 minutes.
3. **Seal and Store the Gel:** Seal the gel with wet paper (Running Buffer or water) and store it in the refrigerator if not used immediately.
4. **Prepare Samples:** Mix 10 µL of Sample Buffer (3X) with 20 µL of each protein sample.
5. **Boil Samples:** Boil the samples at 95ºC for 5 minutes to denature proteins.
6. **Load Samples:** Apply the boiled samples into the wells of the gel.
7. **Run the Gel:** Place the gel into the electrophoresis apparatus, fill with \*\*1X Running Buffer (0.5 mM Tris base, 192 mM glycine, 0.1% SDS, pH 8.3)\*\*, and run at 120 V for approximately 60 minutes. Monitor for bubble formation.
8. **Dye the Gel:** Once electrophoresis is complete, stain the gel with Coomassie Brilliant Blue or another appropriate stain until protein bands are visible.
9. **Unstain the Gel:** Remove excess dye by soaking the gel in destaining solution until clear bands are visible.
10. **Photograph and Analyze:** Photograph the gel using a gel documentation system and analyze the protein bands.

## Protein Expression - Barbie1/BaCBM2 - Mad10

#### Materials

* LB
* Competent Cells Expressing BARBIE or CBM Mad10 (BL21(DE3))
* Flasks
* Antibiotic (Ampicillin)
* Shaker and Incubator
* Spectrophotometer and Cuvette
* Centrifuge
* pH Meter
* Freezer

#### Steps

1. **Pre-inoculum:** Take the appropriate amount of LB media, add Ampicillin (100 ug/uL final concentration), and add 200 µL pre-inoculum cells per 10 mL LB.
2. **Grow Cells:** Incubate the culture in a shaking incubator at 37°C, 220 rpm overnight.
3. **Inoculum Preparation:** Add 10 mL of pre-inoculum to 500 mL LB supplemented with the proper antibiotic. Grow at 37°C, 220 rpm until OD600 reaches 0.8.
4. **Induce Protein Expression:** Add 1 mM IPTG to the culture and incubate at 37°C for 3 hours.
5. **Harvest Cells:** Centrifuge the culture at 5000 g for 15 minutes at 4°C the next day.
6. **Store Pellet:** Store the cell pellet at -80°C until further processing.

## Protein Expression - Barbie1/BaCBM2 - Cys

#### Materials

* LB
* Competent Cells Expressing BARBIE or CBM Cys (BL21(DE3))
* Flasks
* Antibiotic (Kanamycin, 30 µg/mL final concentration)
* Shaker and Incubator
* Spectrophotometer and Cuvette
* Centrifuge
* pH Meter

#### Steps

1. **Pre-inoculum:** Take the appropriate amount of LB media, add Kanamycin (30 µg/mL final concentration), and add 5 µL competent cells per 5 mL LB.
2. **Grow Cells:** Incubate the culture in a shaking incubator at 30°C, 220 rpm overnight.
3. **Inoculum Preparation:** Add 5 mL of pre-inoculum to 500 mL LB supplemented with the proper antibiotic. Grow at 37°C, 220 rpm until OD600 reaches 0.8.
4. **Induce Protein Expression:** Add 0.3 mM IPTG to the culture and incubate overnight.
5. **Harvest Cells:** Centrifuge the culture at 5000 g for 15 minutes at 4°C the next day.
6. **Store Pellet:** Store the cell pellet at -80°C until further processing.

## Protein Expression - Spidroin

#### Materials

* LB
* Competent Cells Expressing Spidroin (BL21(DE3)pRARE2)
* Flasks
* Antibiotics (Kanamycin and Chloramphenicol, 30 µg/mL each final concentration)
* Shaker and Incubator
* Spectrophotometer and Cuvette
* Centrifuge
* pH Meter

#### Steps

1. **Pre-inoculum:** Take the appropriate amount of LB media, add Kanamycin and Chloramphenicol (each 30 µg/mL final concentration), and add 20 µL competent cells per 1 mL LB.
2. **Grow Cells:** Incubate the culture in a shaking incubator at 30°C, 220 rpm overnight.
3. **Inoculum Preparation:** Add 5 mL of pre-inoculum to 500 mL LB supplemented with the proper antibiotics. Grow at 37°C, 220 rpm until OD600 reaches 0.8.
4. **Induce Protein Expression:** Add 1 mM IPTG to the culture and incubate at 20°C overnight.
5. **Harvest Cells:** Centrifuge the culture at 5000 g for 15 minutes at 4°C the next day.
6. **Store Pellet:** Store the cell pellet at -80°C until further processing.

## Protein Expression - Nt-CBM/Nt-Barbie1

#### Materials

* LB
* Competent Cells Expressing Nt-CBM/Nt-Barbie1 (BL21(DE3)pRARE2)
* Flasks
* Antibiotics (Kanamycin and Chloramphenicol, 30 µg/mL each final concentration)
* Shaker and Incubator
* Spectrophotometer and Cuvette
* Centrifuge
* pH Meter

#### Steps

1. **Pre-inoculum:** Take the appropriate amount of LB media, add Kanamycin and Chloramphenicol (each 30 µg/mL final concentration), and add 1 µL competent cells per 1 mL LB.
2. **Grow Cells:** Incubate the culture in a shaking incubator at 30°C, 220 rpm overnight.
3. **Inoculum Preparation:** Add 5 mL of pre-inoculum to 500 mL LB supplemented with the proper antibiotics. Grow at 37°C, 220 rpm until OD600 reaches 0.8.
4. **Induce Protein Expression:** Add 1 mM IPTG to the culture and incubate for 4 hours.
5. **Harvest Cells:** Centrifuge the culture at 5000 g for 15 minutes at 4°C the next day.
6. **Store Pellet:** Store the cell pellet at -80°C until further processing.

## Protein Affinity Purification with Nickel - HisTrap\*

#### Materials

* AKTA Purification System
* Nickel Column (HisTrap HP 5 mL)
* Superloop (to inject sample)
* Sonicator
* Centrifuge
* Buffer A and Buffer B (composition depends on your protein and objective)

#### Steps

1. **Prepare Cell Lysate:**
   * Centrifuge cell pellet in 40 mL Buffer A.
   * Add the following to the resuspension for enhanced lysis and protease inhibition:
     + 1 mM PMSF (serine protease inhibitor)
     + 50 µg/mL Lysozyme (to break down the cell wall)
     + 20 U DNase Turbo (to degrade DNA and reduce viscosity)
     + 100 µM DTT (to prevent oxidation of cysteines and maintain reducing conditions)
     + 40 µL Leupeptin (1,000x stock concentration, a protease inhibitor)
   * Gently mix the suspension and incubate for 30 minutes at 4°C with constant agitation to facilitate lysis.
   * Sonicate the resuspended cells for 10 minutes using the following parameters:
     + 4 seconds ON, 4 seconds OFF pulse cycle to prevent overheating.
     + Amplitude set to 40% to ensure efficient cell disruption.
     + Carry out sonication on ice or in a cold environment to avoid heat-induced protein denaturation.
   * Centrifuge the lysate at 17,000 x g for 1 hour at 4°C to separate the soluble fraction from cellular debris.
   * Transfer the supernatant (soluble protein fraction) to a new tube without disturbing the pellet.
   * Filter the supernatant through a 0.45 µm syringe filter to remove any remaining particulates and ensure a clear solution for column loading.
2. **Set Up AKTA Purification:**
   * Equilibrate the HisTrap column with Buffer A.
   * Load the filtered supernatant containing the protein into the Superloop for loading onto the column.
   * Allow the protein to bind to the column in Buffer A.
   * Perform several washes with Buffer A to remove unbound proteins and impurities.
   * Elute the His-tagged protein using Buffer B containing 300-500 mM imidazole to displace the protein from the nickel resin.
   * Collect the eluted fractions and analyze the protein content by UV absorbance at 280 nm or SDS-PAGE.
   * Pool the fractions containing the desired Barbie-Cys protein.
   * Concentrate the eluted protein if necessary and store at -80°C for future experiments.

[](https://video.igem.org/download/streaming-playlists/hls/videos/de0d2587-ae42-4f32-83f6-d9a9cbf8741b-1028-fragmented.mp4)

## Gel-filtration Chromatography

#### Materials

* AKTA Purification System
* Superdex Column (200)
* Centrifuge
* GF Buffer (composition depends on your protein and objective; no Imidazole)

#### Steps

1. **Prepare Lysate:** Centrifuge the affinity-purified supernatant to remove any remaining debris.
2. **Load the Column:** Inject the clarified lysate into the Superdex column.
3. **Wash the Column:** Wash the column with more than one column volume of GF Buffer to ensure the removal of unbound proteins.
4. **Collect Fractions:** Collect the desired protein peaks based on the elution profiles.
5. **Final Steps:** Combine and concentrate the fractions containing your target protein as needed.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block cover\_image %}
![Results Cover](https://static.igem.wiki/teams/5396/capas/results.webp)
{% endblock %}
{% block page\_content %}

This section presents the results from the production, purification, and characterization of the project parts. To enhance clarity, the results are organized into three distinct DBTL cycles followed during the project's execution.

Briefly, our most important scientific achievements are:

#### Cycle 1

1- Production and purification of the plastic-binding protein BaCBM2-RFP-3xMad10, a Carbohydrate-Binding Module from *Bacillus anthracis* with a three-repeat magnetite-binding tag, Mad10.

2- Production and purification of the plastic-binding protein Barbie1-RFP-3xMad10, a novel protein designed using BaCBM2 as a scaffold with increased affinity for microplastics.

3- Characterization of BaCBM2-RFP-3xMad10 and Barbie1-RFP-3xMad10 using Circular Dichroism (CD) indicated that:

    • The secondary structure absorbance of Barbie1-3xMad10 and BaCBM2-RFP-3xMad10 is similar, as expected from computational simulation evidence;

    • Both proteins exhibit thermal resistance, especially BaCBM2;

    • Both proteins' structures do not change in the presence of plastic nanoparticles;

4- Characterization of BaCBM2-RFP-3xMad10 using Dynamic Light Scattering (DLS) indicated that:

    • By titrating BaCBM2-RFP-3xMad10 in a nanoplastic solution, it was possible to see a coating around the surface of the nanoplastics (protein-corona behavior);

    • Using the Saturation Binding Curve, it was possible to determine that the dissociation constant (Kd) of BaCBM2-RFP-3xMad10 is 1.59 M;

5- Characterization of BaCBM2-RFP-3xMad10 and Barbie1-3xMad10 using Size-Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) indicated that:

    • By analyzing BaCBM2-RFP-3xMad10 with SEC-MALS, three oligomeric states were observed, with the peak of the highest normalized signal indicating that the protein is primarily a monomer, in agreement with Alpha Fold 3 predictions;

    • By analyzing Barbie1-RFP-3xMad10 with SEC-MALS, protein aggregation was detected, with none of the observed peaks aligning with the expected molecular mass of 46.47 kDa;

#### Cycle 2

1- Production and purification of a bioengineered protein derived from spidroin — a spider silk protein — responsible for forming the hydrogel matrix in the filter.

2- Characterization of Nt2RepCt-SpyTag (spidroin) formation using Circular Dichroism (CD):

    • The results demonstrate that increasing temperature leads to the formation of larger hydrodynamic diameter structures, consistent with the formation of possible fibrils;

    • As the temperature rises, the DLS data reveal a rightward shift in particle size distribution, with the 50°C sample showing the largest structures;

3- Characterization of spidroin fibers using Scanning Electron Microscopy (SEM):

    • The SEM revealed an irregular and rough protein hydrogel surface, with signs of fiber formation.

    • Circular structures suggesting potential pore formation were also observed, indicating possible usefulness for particle filtration.

4- Development of a strategy to modify BaCBM2 and Barbie1 with a cysteine tail to enable their application in the [biosensor](https://2024.igem.wiki/cnpem-brazil/hardware) for microplastic detection.

#### Cycle 3

1- Development of a new cloning strategy for the filter proteins, where the fiber-forming domain of spidroin is fused with BaCBM2-Cys/Barbie1-Cys.

2- Production and purification of Nt-Barbie1-Cys and Nt-BaCBM2-Cys proteins.

3- Characterization of Nt-BaCBM2-Cys using Circular Dichroism (CD) indicated that:

    • Nt-BaCBM2-Cys highlights a contrast between Nt2RepCt-SpyTag and BaCBM2-RFP-3xMad10 absorbance, indicating a contribution of each structure to the absorbance;

**Click below to view the detailed reports for each cycle:**

### Cycle 1

In the first cycle of the project, the primary strategy focused on removing microplastics from water treatment facilities using two main components: (1) plastic-binding proteins with high affinity for microplastics and (2) a magnetic tag, Mad10, a three-repeat magnetite-binding tag derived from the magnetotactic bacterium *Desulfamplus magnetovallimortis*. The Mad10 tag would be used to attract the plastic-binding proteins to a magnetic field, allowing them to be removed from the water along with the attached microplastics.

BaCBM2, a Carbohydrate-Binding Module (CBM) from *Bacillus anthracis*, was selected for its strong affinity for plastics. Furthermore, a novel protein was engineered through a [pipeline](https://2024.igem.wiki/cnpem-brazil/model) based on the BaCBM2 structure, modified to enhance its microplastic-binding capabilities, named Barbie1. The results presented here encompass the production, purification, and characterization of the following proteins:

* **Barbie1-RFP-3xMad10**
* **BaCBM2-RFP-3xMad10**

|  |
| --- |
| Barbie1-RFP-3xMad10 |
| Cloning, Transformation & Confirmation  Our biobrick [BBa\_K5396001](https://parts.igem.org/Part%3ABBa_K5396001) encodes the Barbie1 protein, created through reverse engineering of the CBM2 protein from *Bacillus anthracis*. It is also fused with a red fluorescent protein (RFP) and three Mad10 magnetic tags.  The sequence for our Barbie1-RFP-3xMad10 part was chemically synthesized by Genscript and arrived pre-cloned in the pET-15b(+) vector. This vector includes a T7 promoter, lacI, a ribosome binding site (RBS), a 6xHis tag, and a T7 terminator. After the arrival, we proceeded with transforming the plasmid into the *Escherichia coli* BL21(DE3) strain. |

|  |
| --- |
| BaCBM2-RFP-3xMad10 |
| Cloning, Transformation & Confirmation  Our biobrick [BBa\_K5396000](https://parts.igem.org/Part%3ABBa_K5396000) encodes the CBM2 protein from *Bacillus anthracis*. It is also fused to a red fluorescent protein (RFP) and three Mad10 magnetic tags, which facilitate purification using magnetic beads.  The sequence for our BaCBM2-RFP-3xMad10 part was also chemically synthesized by Genscript and arrived pre-cloned in the pET-15b(+) vector. This vector includes a T7 promoter, lacI, a ribosome binding site (RBS), a 6xHis tag, and a T7 terminator. After the arrival, we proceeded with the transformation of the plasmid into the *E. coli* BL21(DE3) strain. |

|  |
| --- |
| Circular Dichroism (CD) |
| A fundamental method for characterizing proteins is using circular dichroism (CD) in the ultraviolet region. The technique is based on the difference of circular polarized light from left to right by optically active entities. Therefore, knowing biomolecules such as proteins have secondary structures, circular dichroism can be very valuable for this task.  Specifically for Barbie1-RFP-3xMad10 and BaCBM2-RFP-3xMad10, CD can give important information once there are beta sheets in their structures. Typically, while the β-sheets present a negative valley around 218 nanometers (nm) and a positive spike around 195 nm, the α-helixes present a negative valley around 222 nm and a positive spike around 190 nm. However, as a limitation of the equipment, the analyzed wavelength (WL) range is between 200 and 250 nm.   Comparison between BaCBM2-RFP-3xMad10 and Barbie1-RFP-3xMad10 structures  ***Comparing the secondary structure absorbance of Barbie1-RFP-3xMad10 and BaCBM2-RFP-3xMad10 highlights its similarity, as expected from the computational simulation evidences***  As Barbie1 was engineered using the BaCBM2 sequence as scaffold, CD can also be very useful to compare both structures and experimentally understand its closeness.  To achieve this, the spectrum acquisition pipeline was followed, with a 2-second integration time for each point across six spectra, all measured at room temperature (20 ºC). From the collected data, it is calculated its average, subtracted from the water absorbance, normalized, and smoothed using a Savitzky-Golay filter.  In Figure 9, the circular dichroism data acquired for both structures are shown. In magenta, Barbie1-RFP-3xMad10 presents a valley similar to the blue curve that represents BaCBM2-RFP-3xMad10. Thus, in the light of the result, it is possible to confirm the structures are very similar.  ![](https://static.igem.wiki/teams/5396/results-nobg/fig9-1-cbmxb1-cd.png)  **Figure 9.** CD acquisition of both Barbie1-RFP-3xMad10 and BaCBM2-RFP-3xMad10 proteins at 20 ºC.   Barbie1-RFP-3xMad10 and BaCBM2-RFP-3xMad10 Heating Ramp  ***From the heating ramp, it was possible to observe some thermal resistance from both plastic binding proteins, specially from BaCBM2-RFP-3xMad10.***  Following on, an essential evaluation for the proteins is the heating ramp, in which we can evaluate how the protein acts in different temperatures and their melting point. For doing so, the same procedure was followed, in which each protein was heated from 20 to 95ºC, as shown in Figure 10. The data acquisition was performed in steps of 5ºC .  Likewise, the curve at 20ºC shown in Figure 10, both proteins presented a similar behavior in different temperatures. From these results, it is evident that both proteins show absorbance in the secondary structure regions, indicating a thermal resistivity.    **![](https://static.igem.wiki/teams/5396/results-nobg/results10.webp)**  **Figure 10.** Heating ramp for Barbie1-RFP-3xMad10 on the left and BaCBM2-RFP-3xMad10 on the right.   Barbie1-RFP-3xMad10 and BaCBM2-RFP-3xMad10 Pontual Spectra  ***By calculating the pontual spectra, on one hand it was notable a constant absorbance for BaCBM2-RFP-3xMad10 through all temperatures, and on the other hand a regime change in the 55 to 60ºC range for Barbie1-RFP-3xMad10***  From the previous results, It is interesting to note that, even after protein engineering, Barbie1-RFP-3xMad10 still maintains its secondary structure. To further explore it, we ran a protein pontual spectra experiment, in which we measured CD absorbance only for 215 nanometers, with each measure made at 1ºC increase.  Figure 11 shows the result of the experiment, which emphasizes the decrease in the CD absorbance for Barbie1-RFP-3xMad10 as already expected from Figure 10. Although Barbie1-RFP-3xMad10 loses part of its absorbance with the increase in temperature, it is not possible to confirm its denaturation. A viable hypothesis is some reduction in the beta-sheets structures.  ![](https://static.igem.wiki/teams/5396/results-nobg/results11.webp)  **Figure 11.** Pontual spectra for Barbie1-RFP-3xMad10 in magenta and BaCBM2-RFP-3xMad10 in blue at 215 nm.  At first glance, this behavior might suggest a drawback in Barbie1’s performance. However, the loss of function at high temperatures could **actually be advantageous for designing the water filter**. Once the filter becomes saturated with microplastics, heating the system to high temperatures would cause Barbie1-RFP-3xMad10 to partially lose its structure. Since the protein’s function is tied to its structure, this loss of affinity would enable the release of microplastics from the filter allowing for their removal and making it also possible to **reuse the Barbie1-RFP-3xMad10 protein in future filtration cycles.**   Heating and Cooling Barbie1-RFP-3xMad10 Ramp  ***By heating*** **Barbie1-RFP-3xMad10*****, it was possible to see the protein loses part of its conformation in the 55 to 60ºC range, but does not recover it in the cooling ramp***  With this possibility in our sights, the following experiment is heating and cooling the protein, in a way we can observe how its structures will behave. Illustrated in Figure 12, the plot was rotated for better visualizing the CD absorbance in function of temperature variation. From left to right, it is possible to firstly see how Barbie1-3xMad10 behaves when heated and then cooled.  As a result, protein’s CD absorbance in the cooling ramp still presented a signal, indicating it maintained its structure and a consequently renovelation of the possible denoveled parts.  ![](https://static.igem.wiki/teams/5396/results-nobg/fig12-1-ramp-cool-heat-b1.png)  **Figure 12.** Heating and cooling ramp CD absorbance of Barbie1-RFP-3xMad10.   Specific Wavelength Analysis  ***As expected from previous results, it was notable through specific wavelengths analysis the regime change in protein absorbance after heating above 55ºC that is not restored***  By focusing our analysis on the 210 nm and 222 nm wavelengths, which correspond to the absorbance region of secondary structures, we can gain a clearer understanding of the variability. In Figure 13, the upper graphs correspond to the 210 nm wavelength, while the lower graphs correspond to the 222 nm wavelength.  A dashed gray line representing the average initial absorbance of Barbie1-RFP-3xMad10 was added to emphasize the loss of the original absorbance. Both wavelengths exhibit an interesting behavior, showing a significant shift between 55 and 60 ºC, which may indicate a critical point for the protein. The cooling graphs clearly demonstrate how the protein loses part of its original signal, suggesting a conformational change due to the temperature variation.  For future adaptation of Barbie1-RFP-3xMad10, it is essential to assess and design the protein in a way it can be further reused for multiple filtration cycles.   ![](https://static.igem.wiki/teams/5396/results-nobg/results13.webp)  **Figure 13.** Two specific wavelengths, 210 nm above and the 222 nm below, plots for the heating and cooling ramp for Barbie1-RFP-3xMad10.  Barbie1-RFP-3xMad10 Melting Temperature  ***With the Barbie1-RFP-3xMad10 heating ramp at 222 nm wavelength, it was possible to fit the Boltzmann curve to find the protein’s melting temperature***  By analyzing a specific wavelength, it is possible to note the curve’s behavior, which is usual for a protein denaturation. Thus, a viable way to uncover the protein’s melting temperature is by fitting the Boltzmann curve in the collected data, which is given by:  ![](https://static.igem.wiki/teams/5396/results/equations/equation-results-2.webp)  Where   * A1 is the protein’s native state; * A2 is the completely unfolded protein’s state; * Tm is the protein’s melting temperature; * k is a constant that represents the curve inclination.   After fitting the curve in Figure 14, the melting point found for Barbie1-RFP-3xMad10 is approximately 58.81ºC. This information is fundamental for building the filter, since it represents stability in a higher temperature. In particular, it is known that in water purifiers the temperatures do not exceed 30 to 35ºC, making the protein appropriate to the system.   ![](https://static.igem.wiki/teams/5396/results-nobg/results14.webp)  **Figure 14.** Boltzmann fit in the absorbance values of Barbie1-RFP-3xMad10 in a heating ramp.   CD Spectrum Before and After Ramp  ***After heating the protein, there is notable regime change for both Barbie1-RFP-3xMad10 and BaCBM2-RFP-3xMad10, which may indicate a contrast in the initial and final structures***  The final test for evaluating BaCBM2-RFP-3xMad10 and Barbie1-RFP-3xMad10 in different temperatures was measuring them before and after the heating ramp. As plotted in Figure 15, an interesting result can be seen: although BaCBM2-RFP-3xMad10 protein does not lose its secondary structure absorbance as much as Barbie1-RFP-3xMad10 does, there is a notable difference in both of them before and after heating.  As shown, there is a visible contrast in the curve shift, just as in the secondary structure valley. In general, although the absorbance maintains a similar behavior throughout the spectrum, this difference in the initial and final structure may indicate a protein conformational change.  ![](https://static.igem.wiki/teams/5396/results-nobg/results15.webp)  **Figure 15.** On the left, it is shown the Barbie1-RFP-3xMad10 absorbance before and after the heating ramp. On the right, it is shown the BaCBM2-RFP-3xMad10 absorbance before and after the heating ramp.   Plastic Interaction Analysis  ***Through secondary structures absorbance, it was possible to observe that both Barbie1-RFP-3xMad10 and BaCBM2-RFP-3xMad10 structures do not change in the presence of plastic nanoparticles***  Finally, the last experimental evaluation at CD was with the polystyrene nanoplastics. We used a 100 nanometer particle which was inserted into a solution with both Barbie1-RFP-3xMad10 and BaCBM2-RFP-3xMad10.  Nevertheless, as reported from the computational simulations, we do not expect any conformational change. Since CD is only evaluating the absorbance in secondary structures, there may not happen major differences in the experiment.  Consequently, Figure 16 shows as projected. When each of Barbie1-RFP-3xMad10 and BaCBM2-RFP-3xMad10 spectra are plotted with the nanoparticle solution spectra, minor changes can be observed. Specifically, this contrast might be a result of the polystyrene nanoparticle scattering, creating noise in the measurement.  ![](https://static.igem.wiki/teams/5396/results-nobg/results16.webp)  **Figure 16.** Plastic binding proteins Barbie1-RFP-3xMad10 and BaCBM2-RFP-3xMad10 spectra acquisition with 100 nm polystyrene nanoparticles.  Considering we were not able to assess the plastic-protein binding, further studies might be done. One important form to evaluate this is the infrared spectroscopy, which allows studying the vibrational frequencies of molecules. |

|  |
| --- |
| Dynamic Light Scattering (DLS) |
| As Barbie1 and BaCBM2 proteins are much smaller when compared to microplastics, it is expected to create a protein-corona structure, just as explored in the protein corona formation model. For this reason, we developed a very simple experiment of protein titration and particle size measurement in the Dynamic Light Scattering (DLS), which will be explored in the next sections.   Protein Corona Formation  ***By titrating BaCBM2-RFP-3xMad10 in a nanoplastic solution, it was possible to see the protein-corona behavior***  The experiment result is shown in Figure 17. Although we used a 100 nanometer polystyrene particle, it is important to note that the equipment has a 30 nm standard deviation, which reflects in the plotted result. In an initial moment shown on Figure 17 inset (a), the BaCBM2-RFP-3xMad10 is added and starts to aggregate in the plastic particle, increasing the particle size.  On the other hand, in the final titrations, the protein has aggregated in the whole nanoplastic surface, which makes it stabilize its size, as represented on Figure 17 inset (b). This effect can also be seen in the following points, since there is no size increase.  It is particularly interesting to note the theoretical and experimental comparison. Knowing that the protein has a 2.5 nm width, the expected theoretical particle size was a 5% increase (from 100 nm to 105 nm). On the other hand, the experimental value found for the experiment was an increase tax of 5.05% (from 122.7 to 128.9 nm), showing a great proximity between them.  ![](https://static.igem.wiki/teams/5396/results-nobg/results17.webp)  **Figure 17.** Dynamic Light Scattering of the average particle size in function of the protein titration. On Subfigure (a), it is shown the initial titration state representation and the final state on Subfigure (b).   Dissociation Constant Calculation  ***Using the Saturation Binding Curve, we fitted the dissociation constant for BaCBM2-RFP-3xMad10***  From the protein-corona formation found in the previous section, it is now possible to fit the saturation bind curve, which is often used in the context of studying the affinity between ligands and receptors. In special, it measures the binding capacity, given by:  ![](https://static.igem.wiki/teams/5396/results/equations/equation-results-1.webp)  Where:   * R is the hydrodynamic radius; * [P] is the protein concentration; * R\_max is the maximum possible occupied radius when all binding sites are occupied; * Kd is the dissociation constant.   In the context of studying the plastic binding affinity, uncovering the experimental protein dissociation constant is fundamental. Therefore, fitting the average value of each point as shown in Figure 18, it was possible to uncover BaCBM2-RFP-3xMad10 dissociation constant as **6x10^-12 M**.  ![](https://static.igem.wiki/teams/5396/results-nobg/results18.webp)  **Figure 18.** Saturation binding curve fit in the DLS resulted data. |

|  |
| --- |
| Size Exclusion Chromatography With Multi-angle Static Light Scattering (SEC-MALS) |
| *Size exclusion chromatography with multi-angle light scattering (SEC-MALS) is a technique used to separate and analyze macromolecules based on their mass, size, and shape ¹.*   BaCBM2-RFP-3xMad10 SEC-MALS result  Knowing that the BaCBM2 protein construction 6His-BaCBM2-RFP-3xMAD10 has a 44.86 kDa molecular mass (MM), it is possible to observe three different oligomeric states in the SEC-MALS result shown in Figure 19. With a smaller normalized signal (dRI), it was possible to observe a MM of 180±2 kDa around 32 minutes of elution, which is close to the molecular mass of a 4 units oligomer.  Following on, a second elution was made around 34 minutes with 86.2±0.5 kDa. According to this molecular mass, it is possible to observe a dimer structure of the protein with a higher dRI. Finally, with the highest dRI and a molecular mass of 46.1±0.2 kDa at 37 minutes of elution, it was possible to identify the protein monomer state.  When compared to the other oligomer states, it was notable that this last elution had the lowest standard deviation when compared to the others. As a conclusion, it is possible to confirm that the BaCBM2-RFP-3xMAD10 construction is most likely to be a single unit protein. This is an interesting result when we compare with the Homo-Oligomer result obtained in the dry lab using Alpha Fold 3, which also indicates a monomer state for the BaCBM2 protein.   ![](https://static.igem.wiki/teams/5396/results-nobg/results19.webp) **Figure 19.** SEC-MALS result of BaCBM2-RFP-3xMAD10.   Barbie1-RFP-3xMad10 SEC-MALS result  After purifying Barbie1-RFP-3xMad10, SDS-PAGE analysis revealed that the sample was eluted along with other *E. coli* proteins. To better characterize the sample and improve its purity, a 30 kDa concentrator was used to separate the larger proteins from the smaller ones. With the sample more concentrated, we proceeded with SEC-MALS analysis. Unlike the results observed for BaCBM2-RFP-3xMad10, the SEC-MALS outcome for Barbie1-RFP-3xMad10 displayed three distinct peaks in the curve (Figure 20). However, none of the three peaks had an estimated molecular mass consistent with the expected mass of Barbie1-RFP-3xMad10 (46.47 kDa).   As noted earlier, during sample preparation for purification, we detected the formation of large aggregates. Additionally, the aggregation score for Barbie1-RFP-3xMad10, calculated by Aggrescan4D, is significantly higher than that of BaCBM2-RFP-3xMad10. Considering the SEC-MALS results along with the SDS-PAGE findings, which confirmed the presence of Barbie1-RFP-3xMad10 in the collected sample, we believe that the peak with a molecular mass of 157.7 kDa corresponds to a protein aggregate.   ![](https://static.igem.wiki/teams/5396/results-nobg/results20.webp) **Figure 20.** SEC-MALS result of 6His-BARBIE1-RFP-3xMAD10.   Barbie1-RFP-3xMad10 x BaCBM2-RFP-3xMad10 SEC-MALS result  The comparison between the two SEC-MALS curves provides new information. Since the y-axis represents the Normalized Signal, it is difficult to compare the curves directly when plotted separately. However, when combined into a single graph, it becomes clear that the peak obtained for BaCBM2-RFP-3xMad10 is much higher than those for Barbie1-RFP-3xMad10, indicating a higher concentration of BaCBM2-RFP-3xMad10 (Figure 21). In fact, when checking the concentrations before the SEC-MALS analysis with the spectrophotometer, the results had already indicated a higher concentration of BaCBM2-RFP-3xMad10 compared to Barbie1-RFP-3xMad10, which is also reflected in the purification chromatograms. This allowed us to perform more characterization analysis of BaCBM2-RFP-3xMad10.   Regarding the difference in production between the two proteins, we propose several hypotheses. The production of Barbie1-RFP-3xMad10 may have been affected by aromatic amino acids, which are abundant in the protein's structure to enhance its affinity with microplastics (since these aromatic residues increase interaction with plastics). The higher number of aggregation-prone residues in Barbie1-RFP-3xMad10, which significantly affects its solubility, could also explain the difficulties in producing this protein. Additionally, protein aggregation can impact cell function and viability and this aggregation tendency also makes the purification process more difficult. In subsequent project cycles, this difference between Barbie1 and BaCBM2 persisted.  ![](https://static.igem.wiki/teams/5396/results-nobg/results21.webp) **Figure 21.** SEC-MALS normalized signal comparison between 6His-BaCBM2-RFP-3xMAD10 and 6His-BARBIE1-RFP-3xMAD10. |

### Cycle 2

In the second cycle of the project, the focus shifted from water treatment facilities to drinking water filtration and domestic water filters due to the feasibility challenges and the large quantity of proteins required for large-scale applications. To facilitate microplastic filtration using conventional water filters, a bioengineered protein derived from spidroin, a spider silk protein, was selected to serve as the filter matrix due to its hydrogel-forming capabilities and advantageous physicochemical properties. The microplastic-binding proteins BaCBM2 and Barbie1 were maintained.

To enable the structuring of the filter, the SpyTag/SpyCatcher system was employed. This technology facilitates the **covalent and irreversible conjugation** of recombinant proteins through a peptide-protein interaction. SpyTag, a small peptide, was fused to spidroin, while SpyCatcher, a complementary protein, was fused to BaCBM2 and Barbie1. Upon interaction, SpyTag and SpyCatcher form a stable covalent bond, integrating the microplastic-binding proteins into the spidroin fibers. This structural integration enhances the efficiency of the filtration process by ensuring the proteins are firmly anchored within the hydrogel matrix formed by spidroin.

Furthermore, new versions of BaCBM2 and Barbie1 were designed with a cysteine residue at the C-terminus to optimize the binding of these proteins to the surface of the [biosensor](https://2024.igem.wiki/cnpem-brazil/hardware) developed for the quantification of microplastics in water.

The results for the proteins are described below:

* **Barbie1-SpyCatcher**
* **BaCBM2-SpyCatcher**
* **NT2RepCt-SpyTag**
* **Barbie1-Cys**
* **BaCBM2-Cys**

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| Barbie1-SpyCatcher |
| Cloning, Transformation & Confirmation  The SpyTag/SpyCatcher system is an protein engineering tool that allows for the specific and irreversible covalent linking of proteins through a short peptide (SpyTag) and a larger protein (SpyCatcher). This system offers high specificity and efficiency, making it ideal for various applications, including protein purification, assembly, and fluorescent labeling.  Our objective was to bind Barbie1 and Nt2RepCt using the SpyTag/SpyCatcher system. To achieve this, Barbie1 was amplified by PCR, while SpyCatcher was chemically synthesized in gBlocks by IDT.  We conducted several assembly attempts via Golden Gate Assembly using the parts: [BBa\_J428341](https://parts.igem.org/Part%3ABBa_J428341), [BBa\_J435350](https://parts.igem.org/Part%3ABBa_BBa_J435350), [BBa\_J435345](https://parts.igem.org/Part%3ABBa_BBa_J435345), Barbie1, SpyCatcher, and [BBa\_J428069](https://parts.igem.org/Part%3ABBa_BBa_J428069). We transformed the plasmids through electroporation into the *E. coli* strain DH5α. However, we could not obtain the correctly assembled plasmid, **which led us to explore a new approach.** |

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| BaCBM2-SpyCatcher |
| Cloning, Transformation & Confirmation  Our objective was to bind BaCBM2 and Nt2RepCt using the SpyTag/SpyCatcher system. To achieve this, BaCBM2 was amplified by PCR, while SpyCatcher was chemically synthesized in gBlocks by IDT.  We conducted several assembly attempts via Golden Gate Assembly using the parts: [BBa\_J428341](https://parts.igem.org/Part%3ABBa_J428341), [BBa\_J435350](https://parts.igem.org/Part%3ABBa_BBa_J435350), [BBa\_J435345](https://parts.igem.org/Part%3ABBa_BBa_J435345), BaCBM2, SpyCatcher, and [BBa\_J428069](https://parts.igem.org/Part%3ABBa_BBa_J428069). We transformed the plasmids through electroporation into the *E. coli* strain DH5α. However, we could not obtain the correctly assembled plasmid, **which led us to explore a new approach**. |

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| Nt2RepCt-SpyTag |
| Cloning, Transformation & Confirmation  Spidroins are the primary proteins that compose spider silk, renowned for their exceptional mechanical properties, including strength, elasticity, and biodegradability. Nt2RepCt has a complex structure that includes both repetitive elements and unique sequences that distinguish it from other spidroins. The N-terminal domain forms amyloid-like fibrils capable of creating hydrogels, which can serve as a platform for protein immobilization.  Our objective was to bind BaCBM2 or Barbie1 and Nt2RepCt using the SpyTag/SpyCatcher system, so our part [BBa\_K5396002](https://parts.igem.org/Part%3ABBa_K5396002) and also has the 13-amino acid SpyTag and was chemically synthesized by IDT as a gBlock.  We assembled the part [BBa\_K5396009](https://parts.igem.org/Part%3ABBa_K5396009) through Golden Gate Assembly using the following parts: [BBa\_J428341](https://parts.igem.org/Part%3ABBa_BBa_BBa_J428341) (linear, digested with BsaI separately and purified from agarose gel), [BBa\_J435350](https://parts.igem.org/Part%3ABBa_BBa_J435350), [BBa\_J435345](https://parts.igem.org/Part%3ABBa_BBa_J435345), [BBa\_K5396002](https://parts.igem.org/Part%3ABBa_K5396002), and [BBa\_J428069](https://parts.igem.org/Part%3ABBa_BBa_J428069). We transformed the plasmids through electroporation into the *E. coli* strain DH5α and confirmed the correct assembly by Sanger sequencing.  Expression and Purification of Nt2RepCt-SpyTag  ***The purification process revealed that a significant portion of Spidroin was present in the flow-through, indicating a need for optimization of the purification buffers and strategy.***  A pre-inoculum was grown overnight at 30ºC before being transferred to a larger LB culture. After reaching the desired optical density, IPTG was added to induce protein expression, which continued overnight at a lower temperature. Following harvest, the cell pellet obtained from 1 liter of culture was resuspended in 40 mL of Buffer A (pH 8.0), supplemented with 1 mM PMSF and 1 mM benzamidine to prevent protease activity.  The resuspended pellet was sonicated to lyse the cells, and the sample was centrifuged at 17,000g for 40 minutes to clarify the lysate. Protein purification was then performed using Immobilized Metal Affinity Chromatography (IMAC) on an ÄKTA system, with a Ni-column equilibrated in Buffer A.  SDS-PAGE analysis revealed the presence of bands corresponding to Spidroin’s expected size (~36 kDa) in the flow-through, indicating incomplete binding to the column (Figure 22).  ![](https://static.igem.wiki/teams/5396/results-nobg/results22.webp)  **Figure 22.** Analysis of Nt2RepCt-SpyTag expression and purification by SDS-PAGE.  In response to these results, we modified Buffer A, by excluding imidazole, and Buffer B (by reducing imidazole concentration) and to change the elution strategy on the ÄKTA system, opting for a direct elution with 100% Buffer B to enhance purification efficiency.   Improved Purification of Nt2RepCt-SpyTag  ***This experiment demonstrated a more efficient purification of Spidroin, with the removal of imidazole from Buffer A playing a crucial role in enhancing binding efficiency during Ni-column purification.***  To improve the purification of Spidroin, which predominantly appeared in the flow-through in our initial attempts, we removed imidazole from Buffer A to enhance protein binding to the Ni-NTA column. Additionally, we adjusted the imidazole concentration in Buffer B and reduced the ÄKTA flow rate to 0.75 mL/min to ensure a gentler elution, aiming to avoid fiber formation during the process (Figure 23).  ![](https://static.igem.wiki/teams/5396/results-nobg/results23.webp)  **Figure 23.** Chromatogram of Nt2RepCt-SpyTag purification using IMAC (Immobilized Metal Affinity Chromatography) on a Ni-column. The peak 2 is highlighted.   SDS-PAGE analysis confirmed the presence of BaCBM2-RFP-3xMad10 in the collected fractions (Figure 8). Despite the UV curve from the ÄKTA system not following a typical pattern, fractions containing the BaCBM2-RFP-3xMad10 protein were successfully identified and collected, highlighting the effectiveness of Triton X-100 in the purification process.  **![](https://static.igem.wiki/teams/5396/results-nobg/results8.webp)****Figure 8.** Analysis of BaCBM2-RFP-3xMad10 expression and purification by SDS-PAGE.   After purification, an SDS-PAGE analysis was performed, confirming that the second peak contained the majority of the Spidroin protein (Figure 24).   ![](https://static.igem.wiki/teams/5396/results-nobg/results24.webp)  **Figure 24.** SDS-PAGE analysis of Nt2RepCt-SpyTag repurification.  To further improve the purification of Nt2RepCt-SpyTag, we proceeded with a second purification step, where peaks 1 and 2 were combined and re-purified using the same process as before. This additional step allowed for a more effective separation of our target protein from other proteins present in the sample, originating from the expression host used. Consequently, we achieved a higher purity of the Nt2RepCt-SpyTag protein (Figure 25).   ![](https://static.igem.wiki/teams/5396/results-nobg/results25.webp)  **Figure 25.** Chromatogram of the Nt2RepCt-SpyTag repurification using IMAC (Immobilized Metal Affinity Chromatography) on a Ni-column. The highlighted peak corresponds to the region with the highest concentration of the target protein.   The SDS-PAGE analysis indicated a reduction in the bands corresponding to other proteins, along with an increase in the band associated with Nt2RepCt-SpyTag (Figure 26).  **![](https://static.igem.wiki/teams/5396/results-nobg/results26.webp)****Figure 26.** SDS-PAGE analysis of Nt2RepCt-SpyTag repurification. The pink circle highlights the band corresponding to Spidroin, confirming its successful expression and purification.  With this confirmation, we proceeded to a third purification step using Size Exclusion Chromatography (SEC) to achieve a more monodisperse sample. SEC effectively separated the sample based on size, with smaller molecules eluting first and larger ones later, resulting in distinct peaks in the chromatogram (Figure27).  ![](https://static.igem.wiki/teams/5396/results-nobg/results27.webp)  **Figure 27.** Chromatogram of the Size Exclusion Chromatography purification of Nt2RepCt-SpyTag. Fractions 20-21-22-23 were collected and used for further characterization of NT2RepCt-SpyTag and subsequent hydrogel formation tests. Based on the SEC results, we selected fractions 20, 21, 22, and 23 for further analysis, as the indicated molecular size was consistent with Nt2RepCt-SpyTag. An SDS-PAGE analysis was then performed on these fractions to confirm the presence and purity of the target protein (Figure 28).  ![](https://static.igem.wiki/teams/5396/results-nobg/results28.webp)  **Figure 28**. SDS-PAGE analysis of Size Exclusion Chromatography fractions from Nt2RepCt-SpyTag purification. The prominent bands in these lanes indicate the presence of Nt2RepCt-SpyTag, with a molecular weight consistent with the expected size of the protein. |

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| Barbie1-Cys |
| Cloning, Transformation & Confirmation  Our biobricks BBa\_K5396004 (basic) / BBa\_K5396008 (composite) encodes the Barbie1 protein, created through reverse engineering of the CBM2 protein from Bacillus anthracis and has an additional amino acid, a cysteine. The cysteine modification allows a strong interaction between the protein and the sensor surface, due to the affinity between the SH group and the Au(111) surface. This increase in interaction with the sensor is essential for amplifying the signal of microplastics in electrochemical measurements.  The Barbie1-Cys is generated by PCR using BBa\_K5396001 as a template. The reverse primer adds the cysteine at the end of the sequence. Our plasmid was assembled using the Golden Gate method with the following parts: BBa\_J428341 (linear, digested with BsaI separately and purified from agarose gel), BBa\_J435350, BBa\_J435345, BBa\_K5396004, and BBa\_J428069. We transformed the plasmids through electroporation into the *E. coli* strain DH5α and confirmed the correct assembly by Sanger sequencing.   Expression and Purification of Barbie1-Cys  ***The purification process revealed that Barbie1-Cys co-eluted with E. coli proteins, highlighting the need to optimize the purification strategy and conditions.***  The pre-inoculum was prepared by growing *E. coli* BL21-pRAREII in LB medium with antibiotics (Kanamycin and Chloramphenicol) overnight. This culture was then transferred into fresh medium, and after reaching an optical density (OD600) of 0.6, IPTG was added to induce protein expression. Following a 3-hour induction period, cells were harvested by centrifugation, and the pellets were stored for subsequent purification.  For the purification process, the cell pellets were resuspended in a buffer with protease inhibitors to prevent protein degradation, sonicated to lyse the cells, and centrifuged to remove debris. Protein elution was attempted using a linear gradient of imidazole-containing buffer. However, a major challenge emerged: Barbie1-Cys co-eluted with the characteristic *E. coli* protein peak, resulting in poor protein separation.  Due to these elution issues, we did not proceed further with the characterization of BARBIE1-Cys, nor testing the sensor with it. Instead, **we shifted focus to the third phase of the project**, which also involves Barbie1-Cys, but fused to the N-terminal of spidroin. In this phase, **we performed additional small-scale induction and expression tests to refine the production and purification processes for improved results**.  ![](https://static.igem.wiki/teams/5396/results-nobg/results29.webp)  **Figure 29.** Chromatogram of the Barbie1-Cys purification using IMAC (Immobilized Metal Affinity Chromatography) on a Ni-column. The highlighted region corresponds to the characteristic peak obtained for the *E. coli* proteins that co-eluted with Barbie1-Cys. |

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| BaCBM2-Cys |
| Cloning, Transformation & Confirmation  Our BBa\_K5396003 (basic) / BBa\_K5396007 (composite) encodes the CBM2 from *Bacillus anthracis* and has an additional amino acid, a cysteine. The cysteine modification allows a strong interaction between the protein and the sensor surface, due to the affinity between the SH group and the Au(111) surface. This increased interaction with the sensor is essential for amplifying the signal of microplastics in electrochemical measurements.  The BaCBM2-Cys was generated by PCR using BBa\_K5396000 as a template. The reverse primer adds the cysteine at the end of the sequence. Our plasmid was assembled using the Golden Gate method with the following parts: BBa\_J428341 (linear, digested with BsaI separately and purified from agarose gel), BBa\_R0010, BBa\_J435345, BBa\_K5396003, and BBa\_J428069. We transformed the plasmids through electroporation into the *E. coli* strain DH5α and confirmed the correct assembly by Sanger sequencing.  However, we did not proceed with the purification, characterization of BaCBM2-Cys, nor testing the sensor with it. Instead, we shifted our focus to the third cycle of our project, which also involves BaCBM2-Cys, but fused to the N-terminal of spidroin. This new phase aims to optimize the production and purification processes, building on the insights gained from our work on the second cycle. |

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| Dynamic Light Scattering (DLS) |
| Dynamic Light Scattering (DLS) Analysis of Nt2RepCt-SpyTag Fibril Formation  ***The results indicate that as the temperature increases, larger hydrodynamic diameter structures emerge, signaling the formation of fibrils.***  Dynamic Light Scattering (DLS) was employed to analyze the behavior of Nt2RepCt-SpyTag after purification and size exclusion chromatography. DLS involves directing a laser beam at the sample and measuring fluctuations in scattered light intensity caused by the Brownian motion of the particles in solution. These fluctuations are dependent on several parameters, including particle size. By applying an autocorrelation function and specific assumptions, the average hydrodynamic diameter of the particles in the sample can be determined.  To investigate the effect of increasing temperature on the sample's structural properties, four aliquots (100 µl each) of the purified Nt2RepCt-SpyTag were prepared at room temperature (25°C). Each aliquot was then subjected to different temperature conditions: 38°C, 42°C, 46°C, and 50°C, for one hour. After this period, the samples were returned to room temperature for 30 minutes, and DLS measurements were conducted immediately afterward. For each aliquot, three complete experimental runs were performed, with each run consisting of 15 measurements lasting 10 seconds each. The timing of the measurements is critical to ensure accurate DLS results, as the fluctuations in scattering are driven by Brownian motion in solution. After obtaining the three sets of data, the results were averaged to compare particle behavior across different temperatures.  The initial hypothesis was that increasing the temperature would destabilize the secondary structure of Nt2RepCt-SpyTag, which would ultimately lead to fibril formation ³. It was expected that as fibrils formed, the average hydrodynamic diameter of the particles would increase.  Figure 30 presents the results (average) obtained for each temperature, while Figure 31 provides a closer look at the region corresponding to larger particle diameters. As the temperature increased, the curves shifted to the right, with the 50°C curve showing the highest intensity in the larger diameter region, indicating that fibril formation occurs with increasing temperature. Additional experiments, including Circular Dichroism and electron microscopy, were performed to corroborate and further substantiate these findings.  ![](https://static.igem.wiki/teams/5396/results-nobg/results30.webp)  **Figure 30.** This figure shows the average hydrodynamic diameter of Nt2RepCt-SpyTag samples measured at various temperatures (38°C, 42°C, 46°C, and 50°C).  ![](https://static.igem.wiki/teams/5396/results-nobg/results31.webp)  **Figure 31.** This figure provides a closer examination of the larger hydrodynamic diameter region from the DLS measurements of Nt2RepCt-SpyTag samples at increasing temperatures. |

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| Circular Dichroism (CD) |
| Nt2RepCt-SpyTag Before and After Heating   ***Comparing Nt2RepCt-SpyTag absorbance before and after a heating ramp, a change in its secondary structure was observed, which might indicate a hydrogel gelation***   With the now synthesized Nt2RepCt-SpyTag spidroin, we evaluated its CD absorbance in two conditions: before and after a heating ramp. It has been previously reported a hydrogel gelation is observed when it is submitted to high temperatures (37 or 60ºC), thus, it is expected that the protein changes its secondary structure when forming this new structure.   For that matter, an interesting result is shown on Figure 32. While on one hand the blue curve represents the protein before ramp, the magenta curve represents it after heating, with a notable contrast in the CD absorbance profile.   Knowing that the original secondary structure absorption happens in a range between 200 and 218 nanometers, it is possible to confirm a conformational change in the protein structure. This result indicates a possible hydrogel gelation.   ![](https://static.igem.wiki/teams/5396/results-nobg/results32.webp)  **Figure 32.** Nt2RepCt circular dichroism absorbance measured before and after heating.   If we analyze the sample appearance shown on Figure 33, we can actually confirm **a new structure formation**! In a different phase, the protein agglomerates, creating a new layer in the solution even without the need of extremely high concentrations. In further expressions, having a higher protein volume and concentration, it will be possible to create a larger structure for water filtering.   ![](https://static.igem.wiki/teams/5396/results-nobg/results33.webp)  **Figure 33.** Hydrogel formation in the Nt2RepCt-SpyTag solution.   Nt2RepCt-SpyTag Heating Ramp  ***By measuring the protein on a heating ramp, a visible conformation change is observed, indicating a non-reversible process and the stability in the hydrogel formation***  To better assess the hydrogel formation, it is now possible to measure a heating ramp for the solution. Heated from 25 to 60ºC with an increased step of 5ºC and with 6 acquisitions for each temperature, the heating ramp result can be visualized on Figure 34.  During the heating process, the protein loses its secondary structure, as indicated by a decrease in CD absorbance within the 200 to 218 nanometer range. In contrast, the protein does not fully regain its secondary structure during the cooling phase. This result demonstrates that only a partial recovery of absorbance occurs, indicating the process is non-reversible.  This is very important information for hydrogel formation. It is fundamental for the hydrogel structure to have great stability in the room temperature, without reversing to the original protein state.   ![](https://static.igem.wiki/teams/5396/results-nobg/results34.webp)  **Figure 34.** Nt2RepCt-SpyTag heating ramp on the left and cooling ramp on the right.   For better visualizing this specific behavior, on Figure 35 the Nt2RepCt-SpyTag circular dichroism is plotted in function of a specific secondary structure absorbance wavelength, that is 210 nanometers. It is evident how the CD absorbance is not recovered at room temperature.   ![](https://static.igem.wiki/teams/5396/results-nobg/results35.webp)  **Figure 35.** Nt2RepCt-SpyTag heating ramp on the left and cooling ramp on the right. |

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| Scanning Electron Microscope |
| Nt2RepCt-SpyTag Hydrogel Scanning Electron Microscope  ***Using a Scanning Electron Microscope, the protein hydrogel surface was captured, showing a great irregularity and possible pore formation***  Now that we were able to properly assess a structure formation after heating Nt2RepCt-SpyTag, it is now possible to better understand its morphology. For doing this, we used a Scanning Electron Microscope (SEM) to capture the hydrogel surface, as shown on Figure 36.  Although the drying process has damaged the hydrogel surface, its topology can still be detected. It is notable that there is a presence of a protein arrangement in the surface, which is rough and irregular. This can be seen by regions that are more bulky and others that are less bulky.   From the linear structures highlighted in more bright white tones, it is possible to relate the structures to a creation of fibers. Moreover, circular structures were created, which can indicate the pore formation and compartments. This can be very useful in the context of filtering particles.   **![](https://static.igem.wiki/teams/5396/results-nobg/results36.webp)**  **Figure 36.** Hydrogel surface captured on a Scanning Electron Microscope. |

### Cycle 3

In the third phase of the project, the strategy was revised once again to simplify the restructuring of the filter. Instead of using the SpyTag/SpyCatcher system for the irreversible conjugation of spidroin and BaCBM2/Barbie1, we decided to fuse the N-terminal of spidroin with BaCBM2/Barbie1. As indicated in the literature, the N-terminal of spidroin is already known to promote hydrogel formation ². This approach allowed us to produce and purify a single protein capable of forming the hydrogel and binding to microplastics.

The results presented below are for:

* Nt-Barbie1-Cys
* Nt-BaCBM2-Cys

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| **Nt-BaCBM2-Cys** |
| Cloning, Transformation & Confirmation  Our biobricks BBa\_K5396005 (basic) / BBa\_K5396010 (composite) encodes the N-terminal of Nt2RepCt fused to our BaCBM2-Cys. The Nt-BaCBM2-Cys was generated through Gibson Assembly, using as a template the previously constructed plasmids BBa\_K5396007 and BBa\_K5396009.  The product of the reaction was transformed into the *E. coli* strain DH5α through electroporation. Plasmid construction was confirmed by Sanger sequencing.   Purification of Nt-BaCBM2-Cys  ***The first attempt for purifying Nt-BaCBM2-Cys proved challenging, as the protein co-eluted with E. coli contaminants. Part of the challenge stemmed from determining an effective protocol for purifying this new fused protein, indicating the need for optimization of the purification strategy to achieve better separation and purity.***  Developing the protocol for induction, expression, and purification of Nt-BaCBM2-Cys was complex, as this new protein, introduced in the third cycle of our project, is a fusion of the N-terminal of spidroin and BaCBM2-Cys. To start, we followed an experimental protocol similar to the one used in the second cycle for Nt2RepCt-SpyTag purification. This approach involved a longer expression period at a lower temperature; additionally, during the purification stage, the buffers remained at pH 8 and were low in salt to avoid premature fiber formation.  The process began by harvesting *E. coli* BL21 cells expressing Nt-BaCBM2-Cys, which were resuspended in 40 mL of Buffer A (20 mM Tris-HCl, pH 8.0). Protease inhibitors PMSF and Benzamidine were added to prevent protein degradation. Sonication was performed in cycles of 4 seconds ON and 4 seconds OFF, at 40% amplitude for 10 minutes. The resulting lysate was centrifuged at 14,000 r.p.m. for 30 minutes at 4°C to separate the soluble fraction containing the target protein from the cell debris.  For protein purification, we used Immobilized Metal Affinity Chromatography (IMAC) on a Ni-column, equilibrated with Buffer A to ensure optimal conditions. The supernatant was loaded onto the column, and a gradient of Buffer B (20 mM Tris-HCl, 300 mM Imidazole, pH 8.0) was applied from 0% to 100% to elute the bound protein. However, during the elution process, only a single peak appeared on the chromatogram, indicating that Nt-BaCBM2-Cys likely co-eluted with *E. coli* proteins, which typically elute at low imidazole concentrations (Figure 37).  ![](https://static.igem.wiki/teams/5396/results-nobg/results37.webp)  **Figure 37.** Chromatogram of the Nt-BaCBM2-Cys purification using IMAC (Immobilized Metal Affinity Chromatography) on a Ni-column. The highlighted region corresponds to the characteristic peak obtained for the *E. coli* proteins that co-eluted with Nt-BaCBM2-Cys.  SDS-PAGE analysis of the eluted fractions confirmed that the protein of interest was present in the *E. coli* peak (Figure 38). This result showed that the purification process was not fully successful in separating Nt-BaCBM2-Cys from the contaminants, highlighting the need for further optimization.   ![](https://static.igem.wiki/teams/5396/results-nobg/results38.webp)  **Figure 38.** SDS-PAGE analysis of Nt-BaCBM2-Cys purification. The pink circle highlights the band corresponding to Nt-BaCBM2-Cys.   Refining the Purification of Nt-BaCBM2-Cys  ***By reducing the flow rate to 0.75 mL/min, we achieved better peak separation and successfully isolated the Nt-BaCBM2-Cys protein during the second purification attempt.***  In this second attempt, the induction and expression steps largely remained unchanged from the first purification protocol. The key change in this protocol was reducing the flow rate to 0.75 mL/min during the purification step, allowing the sample to pass more slowly through the Ni-column. This adjustment enhanced the interaction between the histidine-tagged protein and the column surface, resulting in better peak separation during elution.  Elution was performed using a gradient of Buffer B (20 mM Tris-HCl, 300 mM Imidazole, pH 8.0) at the slower flow rate of 0.75 mL/min, which facilitated improved separation between the *E. coli* protein peak and the desired Nt-BaCBM2-Cys. This time, the Nt-BaCBM2-Cys peak was successfully isolated (Figure 39). Although the yield of purified protein was still insufficient for hydrogel formation, the sample enabled additional characterization, such as circular dichroism.  ![](https://static.igem.wiki/teams/5396/results-nobg/results39.webp)  **Figure 39.** The chromatogram shows the purification of Nt-BaCBM2-Cys using Immobilized Metal Affinity Chromatography on a Ni-column. The highlighted peaks (3 and 4) indicate the fractions with the highest concentrations of Nt-BaCBM2-Cys.  After purification, we performed SDS-PAGE analysis and observed the highest presence of Nt-BaCBM2-Cys in peaks 3 and 4. We concentrated the sample collected from peak 4, successfully discarding the smaller proteins that were also present. With the concentrated sample, we proceeded to the characterization steps.  ![](https://static.igem.wiki/teams/5396/results-nobg/results40.webp)  **Figure 40.** SDS-PAGE analysis of Nt-BaCBM2-Cys purification. The pink circle highlights the band corresponding to Nt-BaCBM2-Cys.  Further optimization of induction and expression is planned to increase protein yields, ensuring that this improved purification protocol can produce larger quantities of Nt-BaCBM2-Cys for future experiments, including tests for hydrogel formation.  Induction and Expression Optimization  ***The SDS-PAGE gel shows a slight increase in Nt-BaCBM2-Cys expression after IPTG addition; however, this expression does not display the characteristic profile expected from a promoter strongly induced by IPTG. This result suggests the possibility of constitutive activity of the AB\_T7\_lacO promoter.***  After the initial attempts to produce and purify the proteins from the third cycle, we proceeded with a small-scale induction and expression test. To begin, we grew a pre-inoculum of Nt-BaCBM2-Cys (BL21) in LB medium until reaching an OD600 of 0.8. At this point, we added 1 mM IPTG to induce protein expression. Aliquots of 1 mL were collected at 0h, 3h, 4h, and 5h after induction. The OD600 was recorded at each collection time to adjust the sample volume for SDS-PAGE analysis.  As shown in Figure 41, a more prominent band appeared at the expected size for Nt-BaCBM2-Cys in the samples taken at 3, 4, and 5 hours. However, the amount of protein in these samples was also higher compared to the 0h sample, which must be considered when interpreting the results. From this analysis, we observed that the promoter used, AB\_T7\_lacO (BBa\_J435350), appears to have constitutive activity, as IPTG addition did not significantly alter protein production in the various expression tests performed throughout the study.  For the proteins from the first cycle, a different promoter was used, and for the same induction test, we observed much stronger bands on the SDS-PAGE gel for BaCBM2-RFP-3xMad10 and Barbie1-RFP-3xMad10. In the case of Nt2RepCt-SpyTag, which used the same promoter as Nt-BaCBM2-Cys, we achieved good production and purification, suggesting that this protein is more suitable for constitutive production.    **![](https://static.igem.wiki/teams/5396/results-nobg/results41.webp)**  **Figure 41:** SDS-PAGE gel showing the expression levels of Nt-Barbie1-Cys and Nt-BaCBM2-Cys at different induction times (0h, 3h, 4h, and 5h) after IPTG addition. The band corresponding to the expected size for Nt-BaCBM2-Cys is indicated by the square brackets. A slight increase in protein expression is observed after 3, 4, and 5 hours of induction, although the profile does not align with the typical response of a strongly IPTG-induced promoter. |

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| **Nt-Barbie1-Cys** |
| Cloning, Transformation & Confirmation  Our biobricks BBa\_K5396006 (basic) / BBa\_K5396011 (composite) encodes the N-terminal of Nt2RepCt fused to our Barbie1-Cys. The Nt-Barbie1-Cys was generated through Gibson Assembly, using as a template the previously constructed plasmids BBa\_K5396008 and BBa\_K5396009.  The product of the reaction was transformed into the *E. coli* strain DH5α through electroporation. Plasmid construction was confirmed by Sanger sequencing.  Purification of Nt-Barbie1-Cys  ***The initial purification attempt for Nt-Barbie1-Cys showed that the protein co-eluted with E. coli contaminants, requiring further optimization of the purification protocol.***  The purification protocol used for Nt-Barbie1-Cys was identical to the one used in the first attempt for Nt-BaCBM2-Cys. The results were also quite similar: the protein expression levels were not as intense as expected, and its elution occurred alongside the characteristic *E. coli* protein peak (Figure 42).  ![](https://static.igem.wiki/teams/5396/results-nobg/results42.webp)  **Figure 42.** The chromatogram shows the purification of Nt-Barbie1-Cys using Immobilized Metal Affinity Chromatography on a Ni-column. The highlighted region corresponds to the characteristic peak obtained for the *E. coli* proteins that co-eluted with Nt-Barbie1-Cys.  SDS-PAGE analysis of the eluted fractions confirmed the presence of Nt-Barbie1-Cys but also highlighted contamination from *E. coli* proteins (Figure 43). Due to this, we decided to conduct small-scale tests of induction and expression before proceeding with additional purification attempts to optimize both protein expression and isolation (see Nt-BaCBM2-Cys Induction and Expression Optimization).  ![](https://static.igem.wiki/teams/5396/results-nobg/results43.webp)  **Figure 43.** SDS-PAGE analysis of Nt-Barbie1-Cys purification. The pink circle highlights the band corresponding to Nt-Barbie1-Cys. |

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| **Circular Dichroism (CD)** |
| With the new Nt-BaCBM2-Cys purified, it is fundamental to properly characterize it. Since it was possible to analyze the secondary structure of both BaCBM2-RFP-3xMad10 and Nt2RepCt-SpyTag, it is now possible to compare each of these structures.  Nt-BaCBM2-Cys Secondary Structures  ***Nt-BaCBM2-Cys highlights a contrast between Nt2RepCt-SpyTag and BaCBM2-RFP-3xMad10 absorbance, indicating a contribution of each structure to the absorbance***  As shown on Figure 44, the Nt-BaCBM2-Cys absorbance is shown on aqua, Nt2RepCt-SpyTag on light blue, and Nt-BaCBM2-Cys as yellow. Although there is a slight shift on Nt-BaCBM2-Cys spectrum when compared to Nt2RepCt-SpyTag, there is a visible similarity between their CD absorbance, specially from 210 to 250 nanometers.  On the other hand, there is a notable contrast on the BaCBM2-RFP-3xMad10 structure, as expected. When analyzing lower wavelengths, there is also an interesting change. Between 205 and 210 nm, Nt-BaCBM2-Cys has a lower absorbance when compared to both Nt2RepCt-SpyTag and BaCBM2-RFP-3xMad10. This behavior might be a result from the BaCBM2-Cys and N-terminus structure combination.   ![](https://static.igem.wiki/teams/5396/results-nobg/results44.webp)  **Figure 44.** Cycle 3 circular dichroism protein comparison of the BaCBM2-RFP-3xMad10 structure, Nt2RepCt-SpyTag, and Nt-BaCBM2-Cys. |

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| **Aggregation Analysis (Aggrescan4D)** |
| ***These results demonstrate that the addition of the MBP tag significantly improved the solubility of Nt-Barbie1-Cys and Nt-BaCBM2-Cys.***  As mentioned in the results of the first cycle, we experimentally observed that Barbie1-RFP-3xMad10 has a propensity to aggregate, complicating the production, purification, and characterization processes. This outcome was also predicted by *in silico* analysis using the Aggrescan4D tool, which predicts the aggregation propensity of proteins in their folded states at different pH values.  In the third cycle, we decided to reanalyze the aggregation characteristics of each individual protein: Nt-BaCBM2-Cys and Nt-Barbie1-Cys. Once again, the results for Barbie1 indicated a significantly higher propensity for aggregation compared to BaCBM2. Based on this outcome, we proposed an improvement for the final cycle of our project (the version we intend to move forward with for the consolidation of our water filter). We introduced a Maltose-Binding Protein (MBP) tag, a common protein expression tag known to significantly enhance the solubility of many proteins. The goal was to improve the production and purification, particularly for Nt-Barbie1-Cys. Alongside the MBP tag, we also added a TEV (Tobacco Etch Virus) protease cleavage site to allow for the removal of the MBP tag after purification. Thus, the newly proposed proteins are MBP-TEV-Nt-BaCBM2-Cys and MBP-TEV-Nt-Barbie1-Cys.  When we reanalyzed the aggregation propensity of these new proteins and compared them with the proteins from the third cycle, the improvement is evident. Figure 45 presents the Total Score, comparing both proteins from the third cycle with the newly proposed proteins. The Total Score is a global indicator of the aggregation propensity/solubility of a protein structure, which depends on the protein size. The more negative the value, the higher the overall solubility. In other words, when comparing Nt-Barbie1-Cys with Nt-BaCBM2-Cys, which have the same size, we observed, as in the first cycle, better solubility (more negative values) for BaCBM2. The same trend was observed when comparing MBP-TEV-Nt-Barbie1-Cys with MBP-TEV-Nt-BaCBM2-Cys.  ![](https://static.igem.wiki/teams/5396/results-nobg/results45.webp)  **Figure 45.** Comparison of the Total Score for aggregation propensity between the proteins from the third cycle (Nt-Barbie1-Cys and Nt-BaCBM2-Cys) and the newly proposed proteins (MBP-TEV-Nt-Barbie1-Cys and MBP-TEV-Nt-BaCBM2-Cys). More negative values indicate higher overall solubility.   Figure 46 allows for a better comparison between the two pairs of proteins, as it shows the Average Score, which is a normalized indicator of the aggregation propensity/solubility of a protein structure. This normalization allows for a comparison of solubility across different protein structures. The more negative the value, the higher the normalized solubility. The results show that, particularly for Barbie1, the addition of the MBP tag significantly improved the protein's solubility. Moreover, the difference between Barbie1 and BaCBM2 decreased considerably, suggesting that the production and purification processes for both proteins will be more similar. **Although this is an** ***in silico*** **result, it indicates a strong possibility of obtaining enough Nt-Barbie1-Cys for the successful production of the hydrogel component in the water filter.**  ![](https://static.igem.wiki/teams/5396/results-nobg/results46.webp)  **Figure 46.** Comparison of the Average Score for aggregation propensity between the third cycle proteins (Nt-Barbie1-Cys and Nt-BaCBM2-Cys) and the newly proposed proteins (MBP-TEV-Nt-Barbie1-Cys and MBP-TEV-Nt-BaCBM2-Cys). |

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| Scanning Electron Microscope |
| Nt2RepCt-SpyTag Hydrogel Scanning Electron Microscope  ***Using a Scanning Electron Microscope, the protein hydrogel surface was captured, showing a great irregularity and possible pore formation***  Now that we were able to properly assess a structure formation after heating Nt2RepCt-SpyTag, it is now possible to better understand its morphology. For doing this, we used a Scanning Electron Microscope (SEM) to capture the hydrogel surface, as shown on Figure 36.  Although the drying process has damaged the hydrogel surface, its topology can still be detected. It is notable that there is a presence of a protein arrangement in the surface, which is rough and irregular. This can be seen by regions that are more bulky and others that are less bulky.   From the linear structures highlighted in more bright white tones, it is possible to relate the structures to a creation of fibers. Moreover, circular structures were created, which can indicate the pore formation and compartments. This can be very useful in the context of filtering particles.   **![](https://static.igem.wiki/teams/5396/results-nobg/results36.webp)**  **Figure 36.** Hydrogel surface captured on a Scanning Electron Microscope. |

### References

1 SEC-MALS for Absolute Molar Mass and Size Measurements. Wyatt Technology <https://www.wyatt.com/solutions/techniques/sec-mals-molar-mass-size-multi-angle-light-scattering.html> (2019).
2 Arndt, T. et al. Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform. Nature Communications 13, (2022).
3 Zhang, Q., Li, M., Hu, W., Wang, X. & Hu, J. Spidroin-Based Biomaterials in Tissue Engineering: General Approaches and Potential Stem Cell Therapies. Stem Cells International 2021, 1–16 (2021).

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