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) 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. 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. 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.

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)

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]