Part:BBa_K2114011

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) ¹ 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 be released by XbaI and PstI and cloned alongside with an appropriate promoter into an integration vector for B. subtilis by 3A assembly ³.

Characterization

This part was used and characterized by [http://2016.igem.org/Team:Freiburg Team Freiburg 2016].

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 (expressing 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

The enzymatic activity of the spore surface-displayed GST was evaluated by a colorimetric GST assay. This assay is based on the GST-catalyzed conjugation of the thiol group of reduced glutathione (GSH) to the GST substrate 1-chloro-2,4-dinitrobenzene (CDNB). The GST-catalyzed reaction produces a dinitrophenyl thioether which can be detected by a spectrophotometer at 340 nm. 25 million modified spores displaying GST were incubated with the substrates and the absorbance was monitored for a time course of 30 min (figure 3 A). The spores displaying GST had a significantly increased change of absorbance at 340 nm in comparison to unmodified wild type spores (figure 3 B). The enzymatic activity was calculated from the linear increase of the absorbance at 340 nm and a turnover of the substrate at 1.5 nmol/ml/min could be determined.

Figure 3: GST assay with GST displaying spores compared to WT. (A) The GST assay was performed with 25 million GST-displaying spores (BBa_K2114011). The absorbance at 340 nm was monitored for a time course of 30 min. Unmodified wild type spores were used as reference (WT). (B) The increase of the absorbance in time is significantly higher for GST displaying spores compared to WT spores.

References

1. GE Healthcare. Glutathione S-transferase (GST) Gene Fusion System. GST Gene Fusion Syst. 1–8 (2009).
2. Hinc, K., Iwanicki, A. & Obuchowski, M. New stable anchor protein and peptide linker suitable for successful spore surface display in B. subtilis. Microb. Cell Fact. 12, 22 (2013).
3. Radeck, J. et al. The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. J. Biol. Eng. 7, 29 (2013).

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