Difference between revisions of "Part:BBa K2332042"

Revision as of 19:14, 23 October 2017

GFP-mutSpyCatcher (Lys31X, for photocaging)

This construct is the fusion of GFP with SpyCatcher with an amber stop codon (TAG) in place of the reactive Lys 31 residue (Lys31X) in SpyCatcher under the control of a constitutive promoter. This construct enables the photocageing and inactivation of SpyCatcher with the introduction of Ne-methyl-L-lysine instead of the reactive lysine required for its binding to SpyTag. 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. For this construct to function, amberless E. coli cells also have to express pyrrolysyl tRNA (tRNA-Pyl (pylT), as well as pyrrolysyl-tRNA synthetase and the UAA, Ne-methyl-L-lysine, must be supplemented in the media. The pyrrolysyl-tRNA synthetase catalyses the acylation of the suppressor tRNACUA with the UAA. During translation, the UAG amber codon in the mRNA is recognized by the acylated tRNACUA and the UAA will be added to the growing polypeptide chain. This construct allows to directly observe through fluorescence microscopy its localization, and most importantly its co-localization with SpyTag variants pre and post photolysis. Particularly for our project, it allows us to quantify its binding onto cell surface displayed Intimin'-SpyTag and the functionality of post-translational light control.

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 723

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 <partinfo>BBa_K2332042 short</partinfo>
-This construct is the fusion of GFP with SpyCatcher with an amber stop codon (TAG) in place of the reactive Lys 31 residue (Lys31X) in SpyCatcher under the control of a constitutive promoter. This construct enables the photocageing and inactivation of SpyCatcher with the introduction of Ne-methyl-L-lysine instead of the reactive lysine required for its binding to SpyTag. 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. For this construct to function, amberless E. coli cells also have to express [https://parts.igem.org/Part:BBa_K1223014 pyrrolysyl tRNA (tRNA-Pyl (pylT)], as well as [https://parts.igem.org/Part:BBa_K1223013 pyrrolysyl-tRNA synthetase] and the UAA, Ne-methyl-L-lysine, must be supplemented in the media. The pyrrolysyl-tRNA synthetase catalyses the acylation of the suppressor tRNACUA with the UAA. During translation, the UAG amber codon in the mRNA is recognized by the acylated tRNACUA and the UAA will be added to the growing polypeptide chain.
+This construct is the fusion of GFP with SpyCatcher with an amber stop codon (TAG) in place of the reactive Lys 31 residue (Lys31X) in SpyCatcher under the control of a constitutive promoter. This construct enables the photocageing and inactivation of SpyCatcher with the introduction of Ne-methyl-L-lysine instead of the reactive lysine required for its binding to SpyTag. 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. For this construct to function, amberless E. coli cells also have to express [https://parts.igem.org/Part:BBa_K2332003 pyrrolysyl tRNA (tRNA-Pyl (pylT)], as well as [https://parts.igem.org/Part:BBa_K1223013 pyrrolysyl-tRNA synthetase] and the UAA, Ne-methyl-L-lysine, must be supplemented in the media. The pyrrolysyl-tRNA synthetase catalyses the acylation of the suppressor tRNACUA with the UAA. During translation, the UAG amber codon in the mRNA is recognized by the acylated tRNACUA and the UAA will be added to the growing polypeptide chain.
 This construct allows to directly observe through fluorescence microscopy its localization, and most importantly its co-localization with SpyTag variants pre and post photolysis. Particularly for our project,  it allows us to quantify its binding onto cell surface displayed Intimin'-SpyTag and the functionality of post-translational light control.