Coding

Part:BBa_K2114011

Designed by: Wladislaw Stroukov   Group: iGEM16_Freiburg   (2016-10-08)
Revision as of 00:20, 20 October 2016 by WladStr (Talk | contribs) (→‎Characterization)


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 GGGGS 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)Verification of surface localization by flow cytometry

Figure 2: Flow cytometry analysis of the surface-displayed fusion construct. Staining of wild type (not transformed) and engineered (transformed with BBa_K2114011) spores with anti-HA antibodies conjugated to Alexa Fluor 647.

The spores of B. subtilis expressing the part BBa_K2114011 were purified by lysozyme treatment to lyse remaining vegetative cells and stained with anti-HA antibodies conjugated to Alexa Fluor® 647 (Cell Signaling Technology®). The antibody could only access surface-localized HA epitopes of the expressed fusion genes and could confirm the successful display of the heterologous protein on the surface of the modified spores resulting in a slight increase of the fluorescence compared to the stained wild type spores. The low amount of fluorescent spores might be attributible to a low display efficiency using the CotG protein as an anchor for surface display.














II) Verification of functionality by GST assay

Figure 3:

The enzymatic activity of the displayed GST was evaluated by a colorimetric GST assay.



















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


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