Difference between revisions of "Part:BBa K2332053"

Latest revision as of 14:36, 25 October 2017

Blue light inducible expression of GFP-SpyTag (Pblind GFP-SpyTag)

This construct encodes the fusion of GFP with SpyTag under the control of Pblind, a blue light inducible promoter. Cells must also express EL222 (BBa_K2332004) for blue light induction.

This part can be used to quantify its binding affinity to SpyCatcher through fluorescence microscopy. 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 this construct in binding affinity assays, one needs to transform E. Coli cells with our Biobrick and expose to blue-light to allow the cells to produce the fusion protein. Then, cells are lysed and the protein purified to be mixed with SpyCatcher construct variants.

This construct allows to directly observe through fluorescence microscopy its localization, and most importantly its co-localization with SpyCatcher variants. Particularly for our project, it allows us to quantify its binding onto cell surface displayed Intimin'-SpyCatcher.

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
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 723

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 <partinfo>BBa_K2332053 short</partinfo>
-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 Intimin'-SpyCatcher construct variants. SpyTag (13 amino acids) and SpyCatcher (138 amino acids, 15 kDa) are protein binding partners that originate from CnaB2 (immunoglobulin-llike collagen adhesin domain) of the FbaB protein, found in the invasive strains of S. pyogenes.
+This construct encodes the fusion of GFP with SpyTag under the control of [https://parts.igem.org/Part:BBa_K2332002 Pblind], a blue light inducible promoter. Cells must also express EL222 ([https://parts.igem.org/Part:BBa_K2332004 BBa_K2332004]) for blue light induction.
+This part can be used to quantify its binding affinity to SpyCatcher through fluorescence microscopy. 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 this construct in binding affinity assays, one needs to transform E. Coli cells with our Biobrick and expose to blue-light to allow the cells to produce the fusion protein. Then, cells are lysed and the protein purified to be mixed with SpyCatcher construct variants.
+This construct allows to directly observe through fluorescence microscopy its localization, and most importantly its co-localization with SpyCatcher variants. Particularly for our project, it allows us to quantify its binding onto cell surface displayed Intimin'-SpyCatcher.
-[[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