Part:BBa_K2332042

GFP-mutSpyCatcher (Lys31X, for photocaging)

This construct is the fusion of GFP with SpyCatcher with an amber stop codon (TAG) in place of the reactive Lys 31 residue (Lys31X) in SpyCatcher under the control of a constitutive promoter. This construct enables the photocaging and inactivation of SpyCatcher with the introduction of Ne-methyl-L-lysine instead of the reactive lysine required for its binding to SpyTag. SpyTag (13 amino acids) and SpyCatcher (138 amino acids, 15 kDa) are protein binding partners that originate from CnaB2 (immunoglobulin-llike collagen adhesin domain) of the FbaB protein, found in the invasive strains of S. pyogenes. 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. This construct allows to directly observe through fluorescence microscopy its localization, and most importantly its co-localization with SpyTag variants pre and post photolysis. Particularly for our project, it allows us to quantify its binding onto cell surface displayed Intimin'-SpyTag and the functionality of post-translational light control.

Usage and Biology

With this construct, only upon light exposure, SpyCatcher is able to bind SpyTag and fluorescence will be observed on the cell surface of cells expressing intimin'-SpyTag. Photocaging for SpyCatcher inactivation is achieved by incorporating a photocaged unnatural amino acid (UAA), Ne-methyl-L-lysine (See figure 1), 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 1: 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 2).

Figure 2: 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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 723