Difference between revisions of "Part:BBa K2332010"

 
 
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<partinfo>BBa_K2332010 short</partinfo>
 
<partinfo>BBa_K2332010 short</partinfo>
  
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To trigger cell adhesion, we decided to make use of the SpyTag and SpyCatcher, two binding partners that form rapid covalent bonds. 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.  
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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. 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. Intimin'-SpyTag and [https://parts.igem.org/Part:BBa_K2332012 Intimin'-SpyCatcher] will be placed under the control of our blue light inducible promoter [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2332002 Pblind] to guide cell structure formation using blue light.
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This biobrick is the fusion of a truncated version of Intimin and SpyTag for the cell surface display of SpyTag.
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===Usage and Biology===
 
===Usage and Biology===
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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.
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[[File:Intimin-fusions.png|thumb|center|500px| Figure 1: Cell surface display of SpyTag and SpyCatcher for cell aggregation.]]
  
 
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Latest revision as of 18:23, 24 October 2017


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. 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. Intimin'-SpyTag and Intimin'-SpyCatcher will be placed under the control of our blue light inducible promoter Pblind to guide cell structure formation using blue light.


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.

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 801
    Illegal NgoMIV site found at 1542
  • 1000
    COMPATIBLE WITH RFC[1000]