Revision as of 19:24, 30 September 2024

T7-Barbie1-Cys

This composite part codes for the Barbie1 protein with an additional amino acid (cysteine), controlled by T7-LacO promoter and is expressed in the presence of IPTG.

Usage and Biology

To design Barbie1 we utilized the BaCBM2 structural model generated by AlphaFold2 to conduct docking assays on six types of plastic: polypropylene (PP), polyethylene (PE), polyethylene terephthalate (PET), nylon (NY), polyvinyl chloride (PVC), and polystyrene (PS). Using Gnina software, we assessed plastic affinity with relaxed parameters, followed by the elimination of overlaps through ChimeraX for visualization and sequence manipulation. A reverse folding process was applied to the docking outputs using LigandMPNN, filtering the original protein set to retain unique positions based on their scores. This approach generated a total of 36,000 sequences (6,000 per plastic type), leading to the identification of an optimized protein sequence named Barbie1, which has the increased ability to bind to plastics when compared to BaCBM2.

The cysteine modification in the sequence 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 Barbie1-Cys was generated by PCR using as template the BBa_K5396001

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

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 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, or sensor testing. Instead, we shifted focus to the third phase of the project, which also involves Barbie1-Cys, but fused to the N-terminal of spidroin: BBa_K5396011

Figure X: 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.

Sequence and Features