Part:BBa_K4430007
GFP-CBD
It is the key part that is responsible for expressing green fluorescent protein (GFP) fusion protein of CBD (GFP-CBD). CBD is from a virulent bacteriophage P108 infecting Staphylococcus aureus (SA). GFP emits green fluorescence under the confocal laser scanning microscope. Under such microscope, green fluorescence on the cell surface of SA after incubation with GFP-CBD proteins indicates specific binding of the proteins to the bacteria. Researches have demonstrated that GFP-CBD can bind strongly to the cell surfaces of SA.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 829
Illegal AgeI site found at 811 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 641
Usage and Results
Plasmid Construction
We designed our functional part GFP-CBD and cloned it into pET28a backbone plasmid chemically synthesized by Tsingke Biotechnology Co., Ltd. Using fusion protein GFP-CBD, we can confirm whether CBD binds strongly to the cell surfaces of SA. We used Gibson assembly method to construct pET28a-GFP-CBD plasmid. The gel electrophoresis results (Figure 1) showed that the GFP-CBD gene was 1330 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-CBD protein (39.8 kDa). As shown in Figure 2, this protein has been successfully expressed and purified.
Specificity of the GFP-CBD
To confirm whether CBD binds strongly to the cell surfaces of SA strains, the specificity of the GFP-CBD was evaluated using common pathogenic bacteria strains as competitors. As we expected, GFP-CBD has a good specificity toward SA (Figure 3)
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