Part:BBa_K4430002
CBD
It is the key part that is responsible for expressing CBD (C-terminal cell-binding domain). CBD is from a virulent bacteriophage P108 infecting Staphylococcus aureus (SA) , which was isolated from hospital sewage. 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 90
Illegal AgeI site found at 72 - 1000COMPATIBLE WITH RFC[1000]
Usage and Results
Plasmid Construction
We designed our functional part CBD and cloned it into pET28a backbone plasmid chemically synthesized by Tsingke Biotechnology Co., Ltd. As mentioned on our design page, in order to separate SA from food samples, CBD was cloned to the plasmid. We used Gibson assembly method to construct pET28a-CBD plasmid. The gel electrophoresis results (Figure 1) showed that the CBD gene was 604 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 CBD protein (12.8 kDa). As shown in Figure 2, this protein has been successfully expressed and purified.
Magnetic separation effect
To determine how efficient magnetic beads-CBD (MBs-gp13) were for capturing SA 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 61.17%, decreasing as bacteria in mix (Table 1).
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