Latest revision as of 02:53, 2 October 2024

T7-Nt-BaCBM2-Cys

This composite part codes for the N-terminal of Spidroin Nt2RepCt fused to our BaCBM2-Cys, controlled by T7-LacO promoter and is expressed in the presence of IPTG.

Usage and Biology

Figure 1. 3D simulation of Nt-BaCBM2-Cys protein.

Nt2RepCt

Spidroins are the primary proteins that compose spider silk. This part contains the N-terminal domain, which is involved in the initial formation of silk fibers and is crucial for the protein's solubility and stability, and is fused to the BaCBM2 protein, that has the ability to bind to plastics. [1]

BaCBM2-Cys

This CBM2, or Carbohydrate-Binding Module 2, is a protein sourced from Bacillus anthracis. It belongs to a broader family of carbohydrate-binding modules that are crucial for the degradation of polysaccharides. These modules are important to break down complex carbohydrates, enabling microorganisms to convert them into usable energy sources. [2]

The cysteine modification allows a strong interaction between the protein and our 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.

Part Generation

The Nt-BaCBM2-Cys was created through PCR amplification and Gibson Assembly utilizing our composite parts:

• BBa_K5396009
• and BBa_K5396010

The product of the reaction was transformed into the E. coli strain DH5α through electroporation. Plasmid construction was confirmed by Sanger sequencing.

This Biobrick consists of the following basic parts:

• BBa_J435350
• BBa_J435345
• BBa_K5396005
• and BBa_J428069

Expression and Purification

Initial Purification

Developing the protocol for induction, expression, and purification of Nt-BaCBM2-Cys was complex, as this new protein 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 2: 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. 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 3. 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

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. Although the yield of purified protein was still insufficient for hydrogel formation, the sample enabled additional characterization, such as circular dichroism.

Figure 4. 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 5. 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 was 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 of Nt-BaCBM2-Cys

After the initial attempts to produce and purify the proteins, 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.

Figure 6: SDS-PAGE gel showing the expression levels of Nt-Barbie1-Cys (BBa_K5396011) 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.

As shown in Figure 6, 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, 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 our 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 (BBa_K5396000) and Barbie1-RFP-3xMad10 (BBa_K5396001). In the case of Nt2RepCt-SpyTag (BBa_K5396009), 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.

Characterization

Circular Dichroism (CD)

Nt-BaCBM2 Secondary Structures

As shown on Figure 7, the BaCBM2 absorbance is shown on aqua, Nt2RepCt on light blue, and Nt-BaCBM2 as yellow. Although there is a slight shift on Nt-BaCBM2 spectrum when compared to Nt2RepCt, 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 structure, as expected. When analyzing lower wavelengths, there is also an interesting change. Between 205 and 210 nm, Nt-BaCBM2 has a lower absorbance when compared to both Nt2RepCt and BaCBM2. This behavior might be a result from the BaCBM2 and Nt structure combination.

Figure 7. Circular dichroism protein comparison of the BaCBM2 structure, Nt2RepCt, and Nt-BaCBM2.

Sequence and Features