Part:BBa_K5396011

T7-Nt-Barbie1-Cys

This composite part codes for the N-terminal of Nt2RepCt fused with Barbie1-Cys protein, controlled by T7-LacO promoter and is expressed in the presence of IPTG.

Usage and Biology

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 BaCBM2 protein, that has the ability to bind to plastics.

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

The Nt-Barbie1-Cys was created through PCR amplification and Gibson Assembly utilizing our composite parts:

• BBa_K5396009
• and BBa_K5396011

The product of the reaction was transformed into the E. coli strain DH5α through electroporation. Plasmid construction was confirmed by Sanger sequencing.

This Biobrick consists of the following basic parts:

• BBa_J435350
• BBa_J435345
• BBa_K5396006
• and BBa_J428069

Expression and purification

Initial Purification

The purification protocol used for Nt-Barbie1-Cys was identical to the one used in the first attempt for Nt-BaCBM2-Cys, BBa_K5396010

The results were also quite similar: the protein expression levels were not as intense as expected, and its elution occurred alongside the characteristic E. coli protein peak.

Figure X. Chromatogram of the Nt-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 Nt-Barbie1-Cys.

SDS-PAGE analysis of the eluted fractions confirmed the presence of Nt-Barbie1-Cys but also highlighted contamination from E. coli proteins. Due to this, we decided to conduct small-scale tests of induction and expression before proceeding with additional purification attempts to optimize both protein expression and isolation.

Figure X. SDS-PAGE analysis of Nt-Barbie1-Cys purification. The pink circle highlights the band corresponding to Nt-Barbie1-Cys .

Induction and Expression Optimization of Nt-Barbie1-Cys

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 743
  
• 1000
  COMPATIBLE WITH RFC[1000]