Coding

Part:BBa_K5396006

Designed by: Alex Johan Mendes Comodaro   Group: iGEM24_CNPEM-BRAZIL   (2024-09-17)


Nt-Barbie1-Cys

This part is the basic part that composes of N-terminal from Spidroin Nt2RepCt, BBa_K5396002, fused to our Barbie1-Cys, BBa_K5396004.

Composite part: BBa_K5396011

Usage and Biology

imagem-2024-10-01-132834364.png

Figure 1. 3D simulation of Nt-Barbie1-Cys.

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 Barbie1 protein, that has the ability to bind to plastics. [1]

Barbie1-Cys

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

This Biobrick was created through PCR amplification and Gibson Assembly utilizing our composite parts:

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 490
  • 1000
    COMPATIBLE WITH RFC[1000]


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