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 2). 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 1). 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

Cells transformed with this construct will express a truncated version of intimin (denoted intimin') fused to SpyTag. As shown in figure 1, Intimin is an outer membrane protein and thus proteins fused to its N termini will be displayed on the cell surface. The reactive aspartate residue (Asp) in SpyTag is able to form a spontaneous and irreversible strong isopeptide bond with the lysine residue (Lys) in SpyCatcher. Thus, when two cell lines expressing either Intimin'-SpyTag or Intimin'-SpyCatcher, are mixed together, cells will aggregate.

Figure 1: Cell surface display of SpyTag and SpyCatcher for cell aggregation.

Placing Intimin'-SpyTag under the control of Pblind along with the constitutive expression of (EL222), will allow the transcriptional control of the cell surface display of SpyTag through blue-light (465nm). Pblind consists of a fusion of the EL222 DNA binding region and the LuxI promoter (see figure 2). The lux box, a 20bp inverted repeat (LuxR and 3-oxo-C6-HSL complex binding region) from the luxI promoter, was replaced with the 18bp DNA binding region of EL222. In the dark, EL222 is inactive as its N-terminal LOV domain represses its DNA-binding C-terminal HTH domain. Upon blue light exposure (465nm), LOV-HTH interaction is released, allowing it to dimerize and bind its binding region, overlapping the -35 region of the luxI promoter. This ultimately results in the recruitment of RNAP and transcriptional activation. Since light can be controlled easily in space, time and degree, this will enable tight spatiotemporal control of gene expression.

Figure 2: Blue light inducible expression system. Under blue light (465nm), the EL222 DNA binding protein dimerises and binds 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)

Applications

Our aim is to develop a precise 3D printing technology that uses light to guide cell adhesion between bacterial cells into 3D structures. Light would also control the production of biomaterials such as PHA or silicates, enabling a final solid structure to be produced from cells. To achieve this, we would combine cells expressing this construct with cells expressing intimin'-SpyCatcher. We would then use light to control cell aggregation with high precision. Similar to lithography, structures would be generated layer by layer from a flat surface by exposing a particular radius with blue light (see figure 3).

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 under blue-light exposure.

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]