Part:BBa_K5396008

T7-Barbie1-Cys

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

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 is 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

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 96
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 30
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 96
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 96
  Illegal AgeI site found at 344
  
• 1000
  COMPATIBLE WITH RFC[1000]