Improve

Overview

This year TJUSLS_China registers a new part BBa_K2933285
which is an improvement of the exsiting part BBa_K2328027
used in our team from previous years.

Function introduction

BBa_K2328027 is a composite part, coding the sequence of smURFP I + Linker A + RBS I + HO-1I. It is a
coexpression system. smURFP (small ultra-red FP) is a type of fluorescent protein. In order to produce
fluoresce, smURFP must be combined with biliverdin (BV).

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. HO-1 is the gene of the precursor of biliverdin. Ho-1 can use the materials of the E.coil
to produce biliverdin.

Fig.1

smURFP in complex with biliverdin

Research value

smURFP and BV produce fluorescence because their excitation and emission wavelengths are longer than other fluorescent proteins, so they exhibit better light penetration under the skin and less light reflected and scattered by their own tissues in mammals, and because of their distance from their own fluorescence wavelengths, their own fluorescence is lower, the contrast of the image is higher, the imaging background is lower, and oxygen and lyase are not needed. Hydrogen peroxide is not less toxic to cells, so it has important research value and application prospect in mammal imaging.

Design

Because the binding rate of smURFP to BV is too low, This year we consider mutating smURFP to increase its binding rate to BV.

According to the literature reviewed, smURFP will slightly increase the binding rate of BV when it mutates at position 56 near the binding site, while according to the mutation of azoflagellin to amino acid at position 56 in other literatures, finally, we selected the mutation of 56 position tyrosine into arginine, and used wild type as the control.

Fig.2

smURFP-Y56R mutant in complex with biliverdin

Check out

By measuring the absorption values of smURFP-BV complex at different nanometer wavelengths, we normalized the first peak (located at the characteristic absorption wavelength of the protein at 280 nm), and compared the integral area ratio of the two absorption peak (BV located at 388nm and BV-smuRFP complexes at 642nm). We found that the ratio of the amount of small molecules BV in the complex to the amount of protein was significantly increased. This shows that the combination of BV and smURFP ability are improved significantly after mutation.

Fig.3

Figure 3：(a)Absorption spectrum of the wild type of BV-mURFP complexes solution.(b)Comparison of absorption spectra of BV-smURFP-Y56R complex solution.

Reference

[1] Coll, R. Mac, Edwards, M.R., Haaksma, C. Some properties of allophycocyanin from a thermophilic blue-green alga [J]. Biophys. Chem. 1978, 8: 369–376.

For more information about the improved part, please visit our part page http://parts.igem.org/Part:BBa_K2933285