Difference between revisions of "Part:BBa K4134066"

Revision as of 07:24, 12 October 2022

BpfA-Atox1-Linker-KanR-loxP-AggC

Improvement

Homologous recombination device design

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

The formation of transmembrane and outer membrane silver nanoparticles and they act as metal shortcuts to bypass redox center-mediated slow electrons.

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.

The density of silver nanoparticles shows the feasibility of our device.

TEM

Figure1(a). Silver nanoparticles under transmission electron microscopy (TEM). There are many quantum dots (silver nanoparticles) attached to the SW-MR-1, while SW-MR-Atox1 has more clusters of silver nanoparticles than it.

Figure1(b). TEM nanoparticle size and elemental analysis Sw-Atox1 AgNPs are relatively small in size, with most nanoparticles of 10-15nm

·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 transmission electron microscopy (TEM)(figure1(a))show that silver nanoparticles are indeed obtained on the cell surface, and compared with the wild type, the size of the recombinant nanoparticles was smaller and the density was higher.

UV-visible Spectroscopy

The characteristic absorbance of silver nanoparticles (410nm) is detected (Figure2). SW-AtoxⅠ has more silver nanoparticles than the control group.

Figure2(ABC). The characteristic absorbance of silver nanoparticles. Silver nanoparticles have their characteristic absorption peak at 410 nm, and it shows that the content of silver nanoparticles in SW-Atox1 reaches the peak in anaerobic conditions.

Sequence and Features