Difference between revisions of "Part:BBa K2114011"

(→‎Usage and Biology)
(→‎Usage and Biology)
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[[File:iG16_schematic_BBa_K2114011.png|350px|thumb|left|Figure 1: Schematic representation of the fusion protein.]]
 
[[File:iG16_schematic_BBa_K2114011.png|350px|thumb|left|Figure 1: Schematic representation of the fusion protein.]]
  
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This part includes glutathione S-transferase (GST) '''[REF: GST from schistosoma]''' fused by an alpha helical linker [2] to the B. subtilis spore coat gene cotG in order to be displayed on the spore surface. The hemagglutinin epitope tag was included in the fusion construct for convenient detection by specific anti-HA antibodies. The cotG gene was amplified from the genome of B. subtilis and the GST was amplified from the expression plasmid pGEX-6P-1 (GE Healthcare). The HA tag and the alpha helical linker were introduced by primer extensions. Both PCR fragments were assembled by Gibson cloning into pSB1C3. The fusion construct can released by XbaI and PstI and cloned alongside with an appropriate promoter into an integration vector for B. subtilis by 3A assembly [3].  
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This part includes glutathione S-transferase (GST) '''[REF: GST from schistosoma]''' fused by a flexible GGGGS linker [2] to the B. subtilis spore coat gene cotG in order to be displayed on the spore surface. The hemagglutinin epitope tag was included in the fusion construct for convenient detection by specific anti-HA antibodies. The cotG gene was amplified from the genome of B. subtilis and the GST was amplified from the expression plasmid pGEX-6P-1 (GE Healthcare). The HA tag and the alpha helical linker were introduced by primer extensions. Both PCR fragments were assembled by Gibson cloning into pSB1C3. The fusion construct can released by XbaI and PstI and cloned alongside with an appropriate promoter into an integration vector for B. subtilis by 3A assembly [3].  
  
  

Revision as of 23:44, 19 October 2016


CotG_G4S_HA_GST

C-terminal fusion of glutathione S-transferase to spore coat gene cotG by a flexible GGGGS linker.


Usage and Biology

Figure 1: Schematic representation of the fusion protein.

This part includes glutathione S-transferase (GST) [REF: GST from schistosoma] fused by a flexible GGGGS linker [2] to the B. subtilis spore coat gene cotG in order to be displayed on the spore surface. The hemagglutinin epitope tag was included in the fusion construct for convenient detection by specific anti-HA antibodies. The cotG gene was amplified from the genome of B. subtilis and the GST was amplified from the expression plasmid pGEX-6P-1 (GE Healthcare). The HA tag and the alpha helical linker were introduced by primer extensions. Both PCR fragments were assembled by Gibson cloning into pSB1C3. The fusion construct can released by XbaI and PstI and cloned alongside with an appropriate promoter into an integration vector for B. subtilis by 3A assembly [3].














Characterization

I) Surface Localization

Figure 2:













II) Functionality

Figure 3:



















Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1315
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 712