Part:BBa_K4134066
BpfA-Atox1-Linker-KanR-loxP-AggC
Improvement
We believe that there are two modes to improve part. One is to optimize the performance of the existing functions of part, and the other is to expand its application scenarios. Here we chose the latter to implement the improvement of BBa_K1795024.
The existing part BBa_K1795024 provides Kanamycin resistance to the cell under the Promoter R0010. In the absence of LacI protein and CAP protein, this part promotes KanR transcription. In the presence of LacI protein and CAP protein, this part inhibits KanR transcription.
Combined with our own project requirements, we hope to engineer it into a reporter gene of homologous recombination. We kept the KanR coding region and removed its initiation codon so that KanR shares the same initiation codon with BpfA. Then Kanamycin resistance can be used as the basis to judge whether the strain is recombinant that displays silver-binding protein. The recombinant is able to grow on the plate with kanamycin, only if the goal sequence and KanR are recombined to the C-terminal of BpfA successfully.
Description
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.
iGEM2022_Nanjing-China Experiment
We proved that this device work in our project.
We recombined Atox1, a silver-binding protein to the C-terminal of a membrane protein of Shewanella. We added silver nitrate to culture medium and the part of silver ions were reduced in situ by the metabolism-generated electron into silver nanoparticles.
TEM
·SW is the control group, it is wild-type Shewanella.
·SW-Atox1 (SA) is the experimental group, and the silver ion-binding protein Atox1 is fused and expressed in the C-terminal of membrane protein BpfA.
The results of TEM show that silver nanoparticles were obtained on the cell surface. Compared with the wild type, the nanoparticle of our strain has a smaller size and higher density.
The results of TEM nanoparticle size and elemental analysis show that the visible nanopartcles cluster are silver, with most nanoparticles of 10-15nm.
UV-visible Spectroscopy
The characteristic absorbance of silver nanoparticles (410nm) is detected (Figure2). SW-AtoxⅠ has more silver nanoparticles than the control group.
The density of silver nanoparticles shows the feasibility of our device. The device recombined our goal sequence(silver-binding protein) to the C-terminal of Shewanella membrane protein. And this device can be used to recombine other small-size protein.
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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