Part:BBa_K4430001
gp15
It is the key part that is responsible for expressing gp15. Gp15 is a receptor binding protein (RBP) from the genome of Vibrio parahaemolyticus (VP) phage GHSM17. We have demonstrated that GFP-gp15 can bind strongly to the cell surfaces of Vibrio parahaemolyticus.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Results
Plasmid Construction
We designed our functional part gp15 and cloned it into pET28a backbone plasmid chemically synthesized by Tsingke Biotechnology Co., Ltd. As mentioned on our design page, in order to separate VP from food samples, gp15 was cloned to the plasmid. We used Gibson assembly method to construct pET28a-gp15 plasmid. The gel electrophoresis results (Figure 1) showed that the gp15 gene was 561 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 gp15 protein (25.4 kDa). As shown in Figure 2, this protein has been successfully expressed and purified.
Magnetic separation effect
To determine how efficient magnetic beads-gp15 (MBs-gp15) were for capturing VP in solution, the capture efficiencies were calculated. Colonies on the plates were counted after incubation for 0.5 h at 37 °C. The numbers of colonies before (nbefore) and after (nafter) magnetic separation were compared to calculate the capture efficiency. The calculated capture efficiency was 97.50%, decreasing as bacteria in mix (Table 1).
| None |