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 == | ||
+ | ===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. | ||
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+ | [[File:Gz1-part-1-16.png|500px|thumb|center|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. | ||
+ | |||
+ | [[File:Gz1-part-1-17.png|500px|thumb|center|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) | ||
+ | |||
+ | [[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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1961
- 1000INCOMPATIBLE 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.
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.
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)