Part:BBa_K811005:Experience

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Applications of BBa_K811005

Tomoki Uchino from [http://2016.igem.org/Team:Kyoto iGEM16_Kyoto] improved and further characterized functionality of this part.
Please see BBa_K1933001, BBa_K1933200, and our [http://2016.igem.org/Team:Kyoto/Description project] for more details.

Confirmation of functionality of BBa_K811005

We used a modified form of BBa_K811005 in Kyoto 2016’s [http://2016.igem.org/Team:Kyoto/Description project] and added further characterization to this part.

First, we constructed INPNC-His-scFv encoding plasmid. scFv we used as a passenger protein to this surface expression domain was anti-Norovirus scFv. It specifically binds to Norovirus(NoV). We transformed it into E. coli strain DH5α to examine the physical interaction between the transformants and NoV-like particles (NoVLPs, the capsid proteins of norovirus). By scanning electron microscopy, we clearly observed NoVLPs bound on the surface of INPNC-His-scFv expressing E. coli cells. This, along with other results in our project show that a protein of interest can be expressed on the surface of E. coli by this INPNC module to capture target molecule(s). This visual demonstration on the function of this part is shown in our [http://2016.igem.org/Team:Kyoto/Description project].

Addition of His-tag to BBa_K811005

[http://2016.igem.org/Team:Kyoto/Description Kyoto 2016] added His-tag to BBa_K811005 and obtained a new part BBa_ K1933001.

This addition introduces two new functions to the fusion protein:

1) We can use anti-His tag antibody to detect their surface expression regardless of passenger protein placed downstream.
We confirmed this with detections of the INPNC-His-(passenger protein) by Western blotting using anti-His tag antibody.

2) INPNC-His-(passenger protein) can be purified using nickel columns.
Taking advantage of this His-tag system, we can isolate our fusion proteins from the insoluble membrane fractions of E. coli cells, after solubilizing with 7M guanidine-HCl. This would allow fusion protein detection even if the protein’s expression levels are low, further strengthening the first function.
We tested and confirmed this function. See our [http://2016.igem.org/Team:Kyoto/Description project] for more details.

User Reviews

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