Part:BBa_K2332051

Intimin'-SpyCatcher (constitutive)

This biobrick is the fusion of a truncated version of Intimin (159kDa), an outer membrane surface protein, and SpyCatcher (138 amino acids, 15 kDa) for the cell surface display of SpyCatcher. To trigger cell adhesion, we decided to make use of the SpyTag and SpyCatcher, two binding partners that form rapid covalent bonds, fused to outer membrane surface proteins. SpyTag (13 amino acids) and SpyCatcher (138 amino acids, 15 kDa) come from CnaB2 (immunoglobulin-llike collagen adhesin domain) of the FbaB protein, found in the invasive strains of S. pyogenes. The reactive lysine in SpyCatcher forms an isopeptide covalent bond with the aspartate residue in SpyTag. We decided to fuse SpyTag and SpyCatcher to outer membrane surface proteins in distinct cell populations that would then aggregate once mixed together. Intimin'-SpyCatcher and Intimin'-SpyTag will be placed under the control of our blue light inducible promoter Pblind to guide cell structure formation using blue light along with our SpyTag variants.

Usage and Biology

Figure 1: Cell surface display of SpyTag and SpyCatcher for cell aggregation. Two cell lines are constitutively expressing a truncated version of intimin, denoted Intimin', fused to SpyCatcher or SpyTag. Intimin is an outer membrane protein and thus proteins fused to its N termini will be displayed on the cell surface. Once the cell lines come together, the reactive lysine in SpyCatcher will form an isopeptide covalent bond with the aspartate residue in SpyTag and cells will aggregate.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 880
  Illegal NgoMIV site found at 1621
  
• 1000
  COMPATIBLE WITH RFC[1000]