Part:BBa_K2114019

GST_HA_G4S_CgeA

N-terminal fusion of glutathione S-transferase to spore crust gene cgeA 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 cgeA 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].

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 688
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1008
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 85