Part:BBa_K2332025

Intimin'-mutSpyCatcher (Lys31X, for photocaging)

This biobrick is the fusion of a truncated version of Intimin (159kDa), an outer membrane surface protein, and an inactive version of SpyCatcher (138 amino acids, 15 kDa) for the cell surface display of inactive SpyCatcher constitutively.

For an immediate induction of cell aggregation and structure formation, we aimed to develop a post-translational light switch in which cells are constitutively expressing an inactive version of SpyCatcher on their cell surface that can only bind cells expressing intimin'-SpyTag upon UV light (365nm) exposure (see Figure 1). SpyTag (13 amino acids) and SpyCatcher (138 amino acids, 15 kDa) form covalent isopeptide bonds and originate from CnaB2 (immunoglobulin-llike collagen adhesin domain) of the FbaB protein, found in the invasive strains of S. pyogenes.

Figure 1: Post-translational light control system for cell aggregation. Cells are constitutively expressing an inactive photocaged version of SpyCatcher on their cell surface that can only bind cells expressing intimin'-SpyTag upon UV light (365nm) photolysis.

Usage and Biology

With this construct, only upon light exposure, SpyCatcher is able to bind SpyTag resulting in precise and rapid control of cell aggregation and structure formation. This is achieved by incorporating a photocaged unnatural amino acid (UAA), Ne-methyl-L-lysine (See figure 2), in place of the reactive lysine in SpyCatcher required for the covalent bond formation with the SpyTag aspartate residue. Upon exposure to UV light (20min, 365nm), the “cage” group in the unnatural photocaged amino acid is cleaved off revealing the native amino acid and a biologically active protein. This approach also adds a layer of bio-containment as cells will only function when externally supplied with UAA.

Figure 2: Photocaged unnatural amino acid (UAA), Ne-methyl-L-lysine

To achieve this, we introduced an amber stop codon (TAG) in place of the reactive Lys 31 residue (Lys31X) in SpyCatcher. For this construct to function, amberless E. coli cells also have to express pyrrolysyl tRNA (tRNA-Pyl (pylT), as well as pyrrolysyl-tRNA synthetase and the UAA, Ne-methyl-L-lysine, must be supplemented in the media. The pyrrolysyl-tRNA synthetase catalyses the acylation of the suppressor tRNACUA with the UAA. During translation, the UAG amber codon in the mRNA is recognized by the acylated tRNACUA and the UAA will be added to the growing polypeptide chain. The orthogonality of this system has shown to work in both E. coli and mammalian cells (see figure 3).

Figure 3: Photocaging mechanism in E. coli cells. Amberless cells must be transformed with plasmids encoding tRNA-Pyl, Pyrrolysyl-tRNA Synthetase and the target protein with an amber codon at the position where the photocaged lysine residue (Ne-methyl-L-lysine) is to be introduced. Figure adapted from Mitra N.

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]