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:

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

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

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):

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

4- Development of a strategy to modify BaCBM2 and Barbie1 with a cysteine tail to enable their application in the biosensor 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:

Barbie1-RFP-3xMad10

Cloning, Transformation & Confirmation

Our biobrick BBa_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.

Optimization of Protein Expression of Barbie1-RFP-3xMad10

The experiment successfully demonstrated significant protein expression of Barbie1-RFP-3xMad10 after IPTG induction, with varying induction times of 3, 4, and 5 hours.

We inoculated E. coli cells expressing Barbie1-RFP-3xMad10 into LB medium with ampicillin, incubating them at 37°C until an OD600 of 0.5 was reached. At this point, 1 mM of IPTG was added to each culture to induce protein expression, and the cultures were left shaking at 37°C for different durations: 3, 4, and 5 hours. 

After harvesting the pellets, we extracted the proteins using a protein extraction buffer and sonication, followed by protein quantification using the BSA assay. The samples were then prepared for SDS-PAGE by denaturing them at 95°C and loaded into the gel for electrophoresis.

The electrophoresis results showed a clear and significant difference in protein expression when comparing the IPTG-induced samples (for 3, 4, and 5 hours) to the control at 0 hours, which had no visible expression bands (Figure 1). However, when comparing the 3, 4, and 5-hour induction times to each other, there was no substantial visual difference in the intensity of the bands, indicating that protein expression was already strong at the 3-hours and remained consistent through the later induction times.

Figure 1. Analysis of protein expression of Barbie1-RFP-3xMad10 over different induction times (0h, 3h, 4h, 5h) via SDS-PAGE.

Expression and Purification of Barbie1-RFP-Mad10

The experiment led to partial purification of Barbie1-RFP-Mad10, but several challenges were encountered during the sample preparation for the ÄKTA purification system.

During the sample preparation, we observed a high level of viscosity, which complicated the purification process. To address this, we centrifuged the sample multiple times and filtered it twice before loading it onto the equipment. Unfortunately, a significant portion of the sample was lost during this additional processing. Despite these challenges, the chromatogram still showed a clear peak corresponding to Barbie1-RFP-Mad10 (Figure 2).

Figure 2. Chromatogram of Barbie1-RFP-Mad10 purification using IMAC (Immobilized Metal Affinity Chromatography) on a Ni-column. The peak corresponding to the Barbie1-RFP-Mad10 protein is highlighted, indicating successful, although partial, purification.

After purification, we sought to understand the cause of the excessive viscosity in our sample. To investigate, we performed a computational analysis using Aggrescan4D (A4D) to predict the aggregation propensities of the protein fold state. The results indicate that Barbie1-RFP-3xMad10 exhibits a higher aggregation propensity compared to BaCBM2-RFP-3xMad10. Figure 3 shows the scores for each residue in both proteins, where residues with negative values are considered "soluble residues", and those with positive values are classified as "aggregation-prone residues". The difference between the two proteins is particularly noticeable in the initial residues, corresponding to BaCBM2 and Barbie1.

Figure 3. Scores for each residue in both proteins (BaCBM2-RFP-3xMad10 and Barbie1-RFP-3xMad10). Residues with negative values are identified as "soluble residues", while those with positive values are considered "aggregation-prone residues”.

Based on this finding, we decided to perform new rounds of expression and purification, this time incorporating detergents to improve protein solubilization. In subsequent cycles, we adapted our strategy to account for the aggregation tendency of Barbie1-RFP-Mad10, aiming to minimize this characteristic and improve the purification process.

BaCBM2-RFP-3xMad10

Cloning, Transformation & Confirmation

Our biobrick BBa_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.

Optimization of Protein Expression of BaCBM2-RFP-3xMad10

The experiment successfully demonstrated significant protein expression for BaCBM2-RFP-3xMad10 after IPTG induction, with the strongest expression observed at 3 and 5 hours.

We inoculated E. coli cells transformed with BaCBM2-RFP-3xMad10 into LB medium supplemented with ampicillin and incubated the cultures at 37°C until they reached an OD600 of 0.5. Once the desired cell density was achieved, 1 mM of IPTG was added to induce protein expression, and the cultures were shaken for 3, 4, or 5 hours, depending on the sample.

After harvesting the pellets, they were resuspended in a protein extraction buffer, sonicated, and incubated on ice. Protein concentration was quantified using the BSA assay, and the samples were prepared for SDS-PAGE. After electrophoresis, the gel was stained and unstained to visualize the protein bands.

The SDS-PAGE results showed a clear expression of BaCBM2-RFP-3xMad10 (Figure 4). The protein bands were more intense at 3 and 5 hours after IPTG induction, indicating higher protein expression at these time points. While expression was observed at 4 hours as well, the bands were not as prominent compared to the 3 and 5-hour time points. This protocol was important to confirm that the tested induction times were sufficient for BaCBM2-RFP-3xMad10 expression.

Figure 4. Analysis of protein expression of BaCBM2-RFP-3xMad10 over different induction times (0h, 3h, 4h, 5h) via SDS-PAGE. 

Expression and Purification of BaCBM2-RFP-Mad10

The experiment successfully demonstrated efficient expression and purification of BaCBM2-RFP-Mad10 using optimized growth and purification protocols.

We initiated the process by growing a pre-inoculum of E. coli BL21(DE3) expressing BaCBM2-RFP-Mad10 in LB medium with ampicillin overnight at 37ºC. The next day, the pre-inoculum was transferred to a larger culture and incubated until it reached the desired cell density. Protein expression was induced by adding IPTG, followed by shaking at 37ºC for 3 hours.

The harvested culture was frozen, and the pellets were later resuspended in a lysis buffer containing Tris-HCl, NaCl, imidazole, lysozyme, and a protease inhibitor cocktail. The cells were lysed through sonication and the debris was removed by centrifugation. The protein was then purified using Immobilized Metal Affinity Chromatography (IMAC) on an ÄKTA system with a Ni-NTA column.

This protocol resulted in the successful purification of BaCBM2-RFP-Mad10, highlighting the efficiency of the combined expression and purification methods (Figure 5).

Figure 5. Chromatogram of BaCBM2-RFP-Mad10 purification using IMAC (Immobilized Metal Affinity Chromatography) on a Ni-column. The peak corresponding to the BaCBM2-RFP-Mad10 protein is highlighted.

To confirm the identity of the protein, an SDS-PAGE analysis was performed, revealing a band consistent with the expected size of BaCBM2-RFP-Mad10 (Figure 6).

Despite successfully purifying a significant amount of BaCBM2-RFP-Mad10, SDS-PAGE analysis revealed that a large portion of the protein remained in the insoluble fraction, which was prepared from the pellet formed during the final centrifugation step before loading the sample onto the ÄKTA system. A similar result was observed for Barbie1-RFP-3xMad10. Consequently, we decided to optimize the extraction process by testing different detergents to improve solubility and protein recovery.

Figure 6. Analysis of BaCBM2-RFP-3xMad10 expression and purification by SDS-PAGE. 

Protein Extraction Efficiency Using Various Detergents

The experiment successfully identified Triton X-100 as the most effective detergent for protein extraction from BL21 cells transformed with the BaCBM2-RFP-3xMad10 gene.

By testing different detergents and conditions, we aimed to evaluate the efficiency of protein extraction from E. coli BL21 cells transformed with the BaCBM2-RFP-3xMad10 gene. The cell pellet was resuspended in a lysis buffer and divided into twelve fractions, each treated with lysozyme and a different detergent, including Sarkosyl, Triton X-100, Tween-20, CHAPS, and glycerol. The fractions were incubated on ice, sonicated, and centrifuged to extract the proteins.

Protein concentrations in the supernatants were measured using the BCA Protein Assay Kit, and the samples were analyzed via SDS-PAGE. The band intensities from the SDS-PAGE gel indicated that Triton X-100 was the most effective detergent for protein extraction, providing the highest yield and quality. Additionally, the use of lysozyme improved protein extraction efficiency compared to sonication alone.

This analysis demonstrates that Triton X-100, combined with lysozyme treatment, is the optimal method for extracting BaCBM2-RFP-3xMad10 protein from E. coli BL21 cells.

Expression and Purification of BaCBM2-RFP-Mad10 with Triton X-100

The experiment successfully demonstrated the use of Triton X-100 to enhance the purification of BaCBM2-RFP-3xMad10.

To improve protein solubility, we resuspended the E. coli pellet in 40 mL of Buffer A with the addition of 1% Triton X-100, along with lysozyme and a protease inhibitor cocktail. Triton X-100 played a crucial role in ensuring effective cell lysis and preventing aggregation during extraction. The mixture was incubated on ice for 30 minutes. After incubation, the sample was centrifuged at 14,000 rpm for 1 hour at 4°C, and the supernatant was filtered using a 0.45 µm filter.

The supernatant was loaded onto a HisTrap column, equilibrated with Buffer A using the ÄKTA system. After loading, the column was washed with 10 column volumes of Buffer A, and protein elution was performed using 5-10 column volumes of Buffer B. Fractions were collected in glass tubes to avoid protein adhesion to plastic surfaces (Figure 7).

Figure 7. Chromatogram of BaCBM2-RFP-Mad10 purification using IMAC (Immobilized Metal Affinity Chromatography) on a Ni-column. The peak corresponding to the BaCBM2-RFP-Mad10 protein 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.

Figure 8. Analysis of BaCBM2-RFP-3xMad10 expression and purification by SDS-PAGE. 

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.

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.

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.

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.

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.

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:

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.

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.

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. 

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.

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:

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.

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.

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.

Figure 20. SEC-MALS result of 6His-BARBIE1-RFP-3xMAD10.

Barbie1-RFP-3xMad10x 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.

Figure 21. SEC-MALS normalized signal comparison between 6His-BaCBM2-RFP-3xMAD10 and 6His-BARBIE1-RFP-3xMAD10.

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, BBa_J435350, BBa_J435345, Barbie1, SpyCatcher, and 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.

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, BBa_J435350, BBa_J435345, BaCBM2, SpyCatcher, and 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.

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 and also has the 13-amino acid SpyTag and was chemically synthesized by IDT as a gBlock. 

We assembled the part BBa_K5396009 through Golden Gate Assembly using the following parts: BBa_J428341 (linear, digested with BsaI separately and purified from agarose gel), BBa_J435350, BBa_J435345, BBa_K5396002, 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 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).

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).

Figure 23. Chromatogram of Nt2RepCt-SpyTag purification using IMAC (Immobilized Metal Affinity Chromatography) on a Ni-column. The peak 2 is highlighted.

After purification, an SDS-PAGE analysis was performed, confirming that the second peak contained the majority of the Spidroin protein (Figure 24).

Figure 24. SDS-PAGE analysis of Nt2RepCt-SpyTag expression and purification.

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).

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). 

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). 

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).

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. 

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.

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.

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.

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.

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). 

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. 

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.

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.

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.

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.

Figure 35. Nt2RepCt-SpyTag heating ramp on the left and cooling ramp on the right.

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 tons, 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.

Figure 36. Hydrogel surface captured on a Scanning Electron Microscope.

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 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).

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. 

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.

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.

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 at 37°C 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-3xMad10and 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.

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.

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 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).

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).

Figure 43. SDS-PAGE analysis of Nt-Barbie1-Cys purification. The pink circle highlights the band corresponding to Nt-Barbie1-Cys .

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.

Figure 44. Cycle 3 circular dichroism protein comparison of the BaCBM2-RFP-3xMad10 structure, Nt2RepCt-SpyTag, and Nt-BaCBM2-Cys.

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.

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.

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).