Part:BBa_K4430010
GFP-TFP
It is the key part that is responsible for expressing green fluorescent protein (GFP) fusion protein of tail fiber protein (TFP). TFP is a receptor binding protein (RBP) from the genome of E. coli phage T7. GFP emits green fluorescence under the confocal laser scanning microscope. Under such microscope, green fluorescence on the cell surface of E.coli after incubation with GFP-TFP proteins indicates specific binding of the proteins to the bacteria. We have demonstrated that GFP-TFP can bind strongly to the cell surfaces of E. coli strains.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2201
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2169
Illegal AgeI site found at 1824
Illegal AgeI site found at 2262 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 641
Illegal BsaI.rc site found at 1263
Usage and Results
Plasmid Construction
We designed our functional part (GFP-TFP) and cloned it into pET28a backbone plasmid chemical synthesized by Tsingke Biotechnology Co., Ltd. Using fusion protein GFP-TFP, we can confirm whether TFP binds strongly to the cell surfaces of E. coli strains. We used Gibson assembly method to construct pET28a-GFP-TFP plasmid. The gel electrophoresis results (Figure 1) showed that the GFP-TFP gene was 2676 bp, as expected. In addition, we confirmed the results by sequencing.
Protein expression test
SDS-PAGE electrophoresis was used to check the expression of GFP-TFP protein (90.9 kDa). As shown in Figure 2, this protein has been successfully expressed and purified.
Specificity of the GFP-TFP
To confirm whether TFP binds strongly to the cell surfaces of E. coli strains, the specificity of the GFP-TFP was evaluated using common pathogenic bacteria strains as competitors. As we expected, GFP-TFP has a good specificity toward E. coli K12 MG1655 (Figure 3)
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