Part:BBa_K2332011

Blue light inducible expression of Intimin'-SpyTag (Pblind Intimin'-SpyTag)

This biobrick is the fusion of a truncated version of Intimin (159kDa), an outer membrane surface protein, and SpyTag (13 amino acids) for the cell surface display of SpyTag under the control of our Pblind, blue light inducible promoter (see Figure 1). 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 (see Figure 2). 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 but only post blue light induction (see Figure 3). For blue-light transcriptional induction, cells must also express EL222 (BBa_K2332004).

Usage and Biology

Figure 1: Blue light inducible expression system. Under blue light, the EL222 DNA binding protein dimerises and bind its binding region within the designed Pblind promoter, overlapping the -35 region of the luxI promoter. This ultimately results in the recruitment of RNAP and transcriptional activation of Intimin'-SpyTag or Intimin'-SpyCatcher. Figure adapted from Jayaraman P. et al. (2016)

Figure 2: Cell surface display of SpyTag and SpyCatcher for cell aggregation. Two cell lines are 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.

Figure 3: Blue light induction of cell aggregation. Representation of two cell lines expressing either intimin’-SpyTag or intimin’-SpyCatcher fusion proteins under the control of PBlind pre and post light induction.

Sequence and Features

Assembly Compatibility:

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