Difference between revisions of "Part:BBa K4430008"

 
 
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<partinfo>BBa_K4430008 parameters</partinfo>
 
<partinfo>BBa_K4430008 parameters</partinfo>
 
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== Usage and Results ==
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===Plasmid Construction===
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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.
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[[File:Gz1-part-1-16.png|500px|thumb|center|Figure 1 Nucleic acid gel electrophoresis results of GFP-TSP.]]
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===Protein expression test===
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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.
 +
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[[File:Gz1-part-1-17.png|500px|thumb|center|Figure 2 Protein SDS-PAGE electrophoresis results of GFP-TSP.]]
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===Specificity of the GFP-TSP===
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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)
 +
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[[File:Gz1-part-1-83.png|500px|thumb|center|Figure 3 GFP-TSP binds strongly to the cell surfaces of SE.]]

Latest revision as of 06:26, 8 October 2022


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.