Difference between revisions of "Part:BBa K2332000"

Latest revision as of 14:16, 25 October 2017

GFP-SpyTag (constitutive)

We designed our GFP-SpyTag Biobrick to test bacterial cell adhesion through fluorescence microscopy. This part produces the GFP-SpyTag fusion protein constitutively, which can then be cell extracted and purified to use in combination with cells expressing our SpyCatcher construct variants. SpyTag (13 amino acids) and SpyCatcher (138 amino acids, 15 kDa) are protein binding partners that form spontaneous irreversible isopeptide bonds between an aspartate residue in SpyTag and a lysine residue in SpyCatcher. These binding partners originate from CnaB2 (immunoglobulin-llike collagen adhesin domain) of the FbaB protein, found in the invasive strains of S. pyogenes.

Figure 1: E. coli cells constitutively expressing GFP-SpyTag

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 705

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 <partinfo>BBa_K2332000 short</partinfo>
-We designed our GFP-SpyTag Biobrick to test bacterial cell adhesion through fluorescence microscopy. This part produces the GFP-SpyTag fusion protein, which we experimentally used in combination with our Intimin-SpyCatcher Biobrick (see experiment).
+We designed our GFP-SpyTag Biobrick to test bacterial cell adhesion through fluorescence microscopy. This part produces the GFP-SpyTag fusion protein constitutively, which can then be cell extracted and purified to use in combination with cells expressing our SpyCatcher construct variants. SpyTag (13 amino acids) and SpyCatcher (138 amino acids, 15 kDa) are protein binding partners that form spontaneous irreversible isopeptide bonds between an aspartate residue in SpyTag and a lysine residue in SpyCatcher. These binding partners originate from CnaB2 (immunoglobulin-llike collagen adhesin domain) of the FbaB protein, found in the invasive strains of S. pyogenes.
-To use the GFP-Spytag, one needs to transform E. Coli cells with our Biobrick and allow the cells to produce the fusion protein. Then, cells are lysed and the protein purified to be used in the experiment.
+[[File:GFP-SpyTag.png|thumb|center|500px| Figure 1: E. coli cells constitutively expressing GFP-SpyTag]]
 <!-- Add more about the biology of this part here