Difference between revisions of "Part:BBa K2933286"

Latest revision as of 02:56, 20 October 2019

smURFP mutation（Tyrosine at 56 position mutates to Arginine）

The mutant of smURFP, Tyrosine at 56 position mutates to Arginine.

Sequence and Features

Usage and biology

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.
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.

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 mutation smURFP(Y56R) gene. (b)Overlap PCR ligation.(c)Enzyme digestion validation.