Part:BBa_K4430008

GFP-TSP

It is the key part that is responsible for expressing green fluorescent protein (GFP) fusion protein of tailspike protein (TSP) (GFP-TSP). TSP is from the genome of Salmonella phage P22. The tailspikes of P22 and its podoviral relatives display modular structures. The smaller N-terminal particle-binding domain (PBD) of the homotrimeric TSP binds the elongated molecule to the phage head. This segment of around 110 amino-acid residues is highly conserved in sequence between different phages of the same morphology. The much larger C-terminal part of TSP mediates binding to the receptor (receptor-binding domain, RBD). GFP emits green fluorescence under the confocal laser scanning microscope. Under such microscope, green fluorescence on the cell surface of SE after incubation with GFP-TSP proteins indicates specific binding of the proteins to the bacteria.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1961
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 641
  

Usage and Results

Plasmid Construction

We designed our functional part GFP-TSP and cloned it into pET28a backbone plasmid chemically synthesized by Tsingke Biotechnology Co., Ltd. Using fusion protein GFP-TSP, we can confirm whether TSP binds strongly to the cell surfaces of SE. We used Gibson assembly method to construct pET28a-GFP-TSP plasmid. The gel electrophoresis results (Figure 1) showed that the GFP-TSP gene was 2719 bp in length, as expected. In addition, we confirmed the results by sequencing.

Figure 1 Nucleic acid gel electrophoresis results of GFP-TSP.

Protein expression test

SDS-PAGE electrophoresis was used to check the expression of GFP-TSP protein (39.8 kDa). As shown in Figure 2, this protein has been successfully expressed and purified.

Figure 2 Protein SDS-PAGE electrophoresis results of GFP-TSP.

Specificity of the GFP-TSP

To confirm whether TSP binds strongly to the cell surfaces of SE strains, the specificity of the GFP-TSP was evaluated using common pathogenic bacteria strains as competitors. As we expected, GFP-TSP has a good specificity toward SE (Figure 3)

Figure 3 GFP-TSP binds strongly to the cell surfaces of SE.