Part:BBa_K5396004
Barbie1-Cys
This BARBIE1 protein is modified with an additional amino acid (cysteine). This enhancement allows it to be effectively utilized in our biosensor technology.
Part Generation
The Barbie1-Cys fragment was generated from a PCR reaction using primers that specifically amplify the linker-Barbie1-linker region of BBa_K5396001
The reverse primer used in this reaction adds a codon that encodes the amino acid cysteine at the end of the sequence.
Usage and Biology
Starting from the BaCBM2 structure model generated by the AlphaFold2 software, we performed docking assays with six types of plastic: polypropylene (PP), polyethylene (PE), polyethylene terephthalate (PET), nylon (NY), polyvinyl chloride (PVC) and polystirene (PS). We made the docking using Gnina software with relaxed parameters to screen many proteins and features for plastic affinity.
Thereafter, the produced overlaps were removed by the docking assays using the ChimeraX software, as well as used for visualization and sequence manipulation. A reverse folding was then performed with the protein output from the docking using the LigandMPNN tool. The original protein set generated from the docking was filtered to maintain just unique positions, considering the associated score , without overlap between them.
By doing that, 6.000 sequences were generated for each ligand, totalizing 6 plastics x 6.000 sequences = 36.000 sequences, as illustrated in Figure 1. The consensus sequence from the 36000 sequence originated our most optimized protein sequence sensitive for several plastics types was named as Barbie1!
Figure 1. Protein-ligand docking representation of the BARBIE1 protein docked with PP, PE, PET, NY, PVC.
The BARBIE1 protein was redocked with the same plastics as before, once more using Gnina software. The result comparing its affinity with BaCBM2 in silico assays performed are described in the barplot of Figure 5. Comparing the predicted affinity between the original and the modified protein, it is notable a substantial increase for all plastics, in particular for PE, PP, and PS, highlighting the effectiveness of the processing pipeline.
Besides the monomers tests, we also wanted to test the affinity using different sizes of plastic in order to guarantee that this could be a valuable parameter to future analysis and experiments. Therefore, the tested ligands were PE and PET plastics with 50 and 25 repeating units, respectively. As a result, the previous behavior at maintaining a higher KD for Barbie1 when compared to BaCBM2 was preserved.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 91
- 1000COMPATIBLE WITH RFC[1000]
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