Difference between revisions of "Part:BBa K2933285"

Revision as of 05:02, 10 October 2019

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

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Sequence and Features

Usage and biology

In this part, we mutated the tyrosine of smURFP at position 56 to arginine.This change led to a significant increase in smURFP's ability to bind to the small molecule BV.
We co-expressed smURFP and Biliverdin-producing protein heme oxidase (Heme Oxygenase-1, HO-1) in E. coli and then purified the smURFP protein that binds BV small molecules. We used wild-type smURFP as a control and measured the absorbance at specific wavelengths of proteins, small molecule BV, and smURFP-BV complexes by ultraviolet spectrophotometer (280 nm, 388 nm, 642 nm, respectively). We normalized the wild type and mutant absorbance at 280 nm. Through data analysis, we found that the ratio of small molecule BV binding to protein in the mutant complex was significantly greater than that of wild type, which significantly demonstrated that the ability of the mutant to bind small molecules BV had been greatly improved.
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.
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. For more information, see the part BBa_K2328027.

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.

Molecular cloning

We used the vector pet-28b-sumo vector to construct our expression plasmid. We converted the plasmid to E. coli BL21(DE3) for expression and purification.

Figure 1. (a)Cloning of wild type smURFP gene and ho-1 gene. (b)Overlap PCR ligation. (c)Enzyme digestion validation.

Figure 2. (a)Cloning of mutation smURFP(Y56R) gene and ho-1 gene.(b)Overlap PCR ligation.(c)Enzyme digestion validation.

Protein expression 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.