Difference between revisions of "Part:BBa K5396001"

Revision as of 18:10, 23 September 2024

Barbie1_RFP_3xMad10

BARBIE1 is a synthetic protein derived from BaCBM2 through a process of reverse engineering. It has the increased ability to bind to plastics when compared to BaCBM2.

The BARBIE1 protein is fused with the red fluorescent protein (RFP)[ ], which exhibits an excitation maximum at 558 nm and an emission maximum at 583 nm. This fusion enhances the visualization of BARBIE1 by fluorescence-based methods.

This part was used as template to construct BBa_K5396004.

Usage and Biology

Metodo de criação da barbie, importancia da rfp e da tag mad10.

Figure 1. AlphaFold 3 3D simulation of Barbie1 protein with miRFP and three Mad10 tags.

Protein Expression and Purification

The sequence for this 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

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 (3, 4, and 5 hours) to the control at 0 hours, which had no visible expression bands. 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-hour mark and remained consistent through the later induction times.

Figura X. Analysis of protein expression of Barbie1-RFP-3xMad10 over different induction times (0h, 3h, 4h, 5h) via SDS-PAGE. Lanes: 1- Ladder (molecular weight marker), 2- Barbie1-RFP-3xMad10 (0h), 3- Barbie1-RFP-3xMad10 (3h), 4- Barbie1-RFP-3xMad10 (4h), 5- Barbie1-RFP-3xMad10 (5h), 6- BLS1 (untransformed control).

Final expression and purification

During the sample preparation, we observed a high level of viscosity, which complicated the 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.

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 indicated that BARBIE1-RFP-Mad10 has a higher aggregation propensity compared to BaCBM2.

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.

Figure X. 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.

Characterization

SEC-MALS

Circular Dichroism (CD)

CD can provide crucial insights due to the presence of beta sheets in their structures. Typically, β-sheets exhibit a characteristic negative band around 218 nanometers (nm) and a positive band near 195 nm, while α-helices show a negative band around 222 nm and a positive band around 190 nm. However, a limitation of the equipment used restricts the analyzed wavelength range to between 200 and 250 nm.

The pipeline acquisition of the spectrum was followed by a 2 seconds of integration for each point in each 6 spectrum calculated 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.

CD acquisition of Barbie1-RFP-3xMad10 protein at 20 ºC.

Sequence and Features