Part:BBa_K4430006
GFP-gp15
It is the key part that is responsible for expressing green fluorescent protein (GFP) fusion protein of gp15 (GFP-gp15). Gp15 is a receptor binding protein (RBP) from the genome of Vibrio parahaemolyticus (VP) phage GHSM17. GFP emits green fluorescence under the confocal laser scanning microscope. Under such microscope, green fluorescence on the cell surface of VP after incubation with GFP-gp15 proteins indicates specific binding of the proteins to the bacteria. We have demonstrated that GFP-gp15 can bind strongly to the cell surfaces of VP.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 641
Usage and Results
Plasmid Construction
We designed our functional part GFP-gp15 and cloned it into pET28a backbone plasmid chemically synthesized by Tsingke Biotechnology Co., Ltd. Using fusion protein GFP-gp15, we can confirm whether gp15 binds strongly to the cell surfaces of VP. We used Gibson assembly method to construct pET28a-GFP-gp15 plasmid. The gel electrophoresis results (Figure 1) showed that the GFP-gp15 gene was 1639 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-gp15 protein (52.4 kDa). As shown in Figure 2, this protein has been successfully expressed and purified.
Specificity of the GFP-gp15
To confirm whether gp15 binds strongly to the cell surfaces of VP, the specificity of the GFP-gp15 was evaluated using common pathogenic bacteria strains as competitors. As we expected, GFP-gp13 has a good specificity toward VP (Figure 3)
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