Part:BBa_K4134067
pUC57mini-BpfA-Atox1-Linker-KanR-loxP-AggC
Description
For detailed design information, you can look up the registry of BBa_K4134066, the device for fusion protein recombination on the membrane protein of S. oneidensis. This linear device is attached to pUC57mini on both ends and cyclized into a plasmid. When introduced to E.coli(DH 5alpha), it amplifies. Moreover, pUC57mini consists of the replication origin, AmpR, and its promoter.
We designed a device for fusion protein recombination. With this device, you can display any small protein to the C-terminus of BpfA, a large membrane protein of S. oneidensis.
Considering the addition of KanR, you can easily confirm whether homologous recombination succeeds or not, since only the recombinants can grow into colonies on the medium supplemented with kanamycin.
To use this device, all you need to do is to replace Atox1 with your destination fragment. We have proved its feasibility by fusing our destination fragment (Atox1) to the C-terminus of BpfA. The recombinants show resistance to kanamycin and enrich silver ions on the cell membrane.
Moreover, there is a flexible linker between your destination fragment and KanR. Linker is an amino acid chain that acts as a link between two fusion proteins and is flexible enough to allow the proteins on both sides to perform their independent functions.
In detail, BpfA stands for the biofilm-promoting protein A, a large surface protein. The 1000bp-long fragment of BpfA and the 1000bp-long fragment of AggC, the downstream genes of BpfA, are attached to our vector for homologous recombination so as to fuse the destination fragment to the C-terminus of BpfA. This design enables the destination gene to be displayed on the surface of S. oneidensis. Considering the large size of BpfA, this fusion expression does nearly no harm to the normal physiological state of S. oneidensis and we have approved it through a biofilm growth test and bacterial density measurement.
The loxP site-specific recombination site regulates the insertion of the destination fragment at the specific location bpfA, ensuring the fusion of it to bpfA through homologous recombination.
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]
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