Difference between revisions of "Part:BBa K2933285"

Revision as of 14:57, 9 October 2019

smURFP（mutation Y56R） + Linker A + RBS I + HO-1 I

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Sequence and Features

Usage

smURFP (small ultra-red FP) is an important part in our group. It is desirable for our BV detection and in-vivo imaging because with it molecule less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow and orange FPs. smURFP can covalently attaches a biliverdin(BV) chromophore without a lyase, and has 642/670 nm excitation - emission peaks, a large extinction coefficient and quantum yield, and photostability comparable to that of eGFP.

Reference

[1] Rodriguez EA,Tran GN , Gross LA, et al. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein .[J].NATURE METHODS,2016:763-769.

Results

We did some experiments on smURFP and BV.

Biology

In order to fluorescence, Site-directed mutation smURFP must be combined with biliverdin (BV) .So we construct the surface display system to make in-vivo imaging come true. To construct the surface display system, the gene of fluorescent protein---smURFP and the gene of the anchoring protein should be connected to the same expression vector. After the recombinant plasmid is transferred to the target bacteria, the fluorescent protein and anchoring protein will express at the same time and become fusion protein, and then the fluorescent protein will be carried to the cell surface by anchoring protein. With the added biliverdin, fluorescent protein will combine with biliverdin and glow on the cell surface.

Construction of expression Vecto

Using one T7 promoter, two RBS structures, simultaneously expressing smURFP and producing BV heme oxidase.

Figure 1. (a)Cloning of smurfp gene and ho-1 gene.(b)Overlap PCR ligation.(c)After digested

Figure 2. (a)Point mutation PCR of smurfp gene.(b)Point mutation Overlap PCR ligation.(c)After digested

Protein production and purification

Figure 3. (a)Separation of peak position from molecular sieves of smURFP and BV complexes.(b)SDS-PAGE

Figure 4. (a)Separation of peak position from molecular sieves of smURFP and BV complexes.(b)SDS-PAGE

Figure 5. (a)Absorption spectrum of smURFP complex solution.(b)Comparison of absorption spectra of smURFP Y56R complex solution.