Difference between revisions of "Part:BBa K5396004"
 
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<partinfo>BBa_K5396004 short</partinfo>
 
<partinfo>BBa_K5396004 short</partinfo>
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<p>
 
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BARBIE1 is a synthetic protein derived from BaCBM2 (BBa_K5396003) through a process of reverse engineering. It has the increased ability to bind to plastics when compared to BaCBM2.</p><p>This BARBIE1 protein is modified with an additional amino acid (cysteine). This enhancement allows it to be effectively utilized in our biosensor technology.</p>
 
  
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===Usage and Biology===
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This Barbie1 protein is modified with an additional amino acid (cysteine).
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<p>The BaCBM2-Cys fragment was generated from a PCR reaction using primers that specifically amplify the linker-BaCBM2-linker region of Bba_BBa_K5396000. The reverse primer used in this reaction adds a codon that encodes the amino acid cysteine at the end of the sequence.</p>
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=Usage and Biology=
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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.
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https://static.igem.wiki/teams/5396/registry/barbie1-3d.png
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'''Figure 1.''' 3D simulation of Barbie1-Cys protein.
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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.
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=Part Generation=
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The Barbie1-Cys fragment was generated from a PCR reaction using primers that specifically amplify the linker-Barbie1-linker region of <partinfo>BBa_K5396001</partinfo>
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The reverse primer used in this reaction adds a codon that encodes the amino acid cysteine at the end of the sequence.
  
 
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Latest revision as of 20:31, 1 October 2024


Barbie1-Cys

This Barbie1 protein is modified with an additional amino acid (cysteine).

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.

barbie1-3d.png

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

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 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.

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 91
  • 1000
    COMPATIBLE WITH RFC[1000]