Part:BBa_K5136035

inpnc-His tag-SpyCatcher Biology INPNC Ice nucleoprotein (INP), an outer membrane protein from Pseudomonas syringae, has been used as a surface anchor in many researches. The truncated version of INP, namely INPNC which contains only the N- and C-terminal portion of INP, has excellent capacity to anchor target proteins on the cell membrane (1). SpyCatcher SpyCatcher is a engineered split fragment of the fibronectin-binding protein (FbaB) in Streptococcus pyogenes, containing 116 residues. SpyTag is a peptide tag of 13 residues developed by previous researchers. SpyCatcher could steadily bind to SpyTag by forming an isopeptide bond (2). Usage We aim to displaying metallothioneins MT2A and MT3 on the surface of E. coli by INPNC, which could improve the adsorption rate of heavy metal ions for the harmless treatment of deinking wastewater. However, it is tricky to verify whether a protein has been successfully displayed on the surface of bacteria. So, we introduce the Spy system. In our project, SpyCatcher is fused with INPNC and SpyTag is fused with GFP. Then, engineered bacteria which express the fused protein INPNC-his tag-SpyCatcher are treated with SpyTag-GFP. By monitoring the fluorescence intensity of bacteria, we can verify if INPNC could anchor target proteins on cell surface. Here, we constructed the composite part (BBa_K5136224) to express INPNC-his tag-SpyCatcher.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 481
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 330
  Illegal XhoI site found at 1089
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 72
  Illegal NgoMIV site found at 405
  Illegal AgeI site found at 429
  
• 1000
  COMPATIBLE WITH RFC[1000]