Part:BBa_K4430003
TSP
It is the key part that is responsible for expressing TSP (tailspike protein) 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).
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 1222
- 1000COMPATIBLE WITH RFC[1000]
Usage and Results
Plasmid Construction
We designed our functional part TSP and cloned it into pET28a backbone plasmid chemically synthesized by Tsingke Biotechnology Co., Ltd. As mentioned on our design page, in order to separate SE from food samples, TSP was cloned to the plasmid. We used Gibson assembly method to construct pET28a-TSP plasmid. The gel electrophoresis results (Figure 1) showed that the TSP gene was 1993 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 TSP protein (62.6 kDa). As shown in Figure 2, this protein has been successfully expressed and purified.
Magnetic separation effect
To determine how efficient magnetic beads-TSP (MBs-TSP) were for capturing SE 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 11.11%, decreasing as bacteria in mix (Table 1).
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