Difference between revisions of "Part:BBa K1055000"

Latest revision as of 18:24, 12 October 2013

mKate is a fluorophor derived from dsRed

mKate is a fluorophor derived from dsRed and is a FRET partner of LssmOrange [BBa_K1055001].

ATTENTION: If you want to make a fusion protein with this part, please refer to our experiences!

Usage and Biology

Characterization mKate

Figure 1. Peller of E.coli Bl21(DE3) cells with expressed mKate. Left side: without UV radiation, right side: with UV radiation

mKate is a red fluorescent protein that we want to use as a acceptor for FRET (Figure 3). According to [http://www.evrogen.com/products/basicFPs.shtml Evrogen] mKate has an excitation maximum at 588 nm and an emission maximum at 633 nm. Figure 2 shows that the excitation maximum is at 579 nm and the emission maximum is 611 nm. The emission maximum is big enough to get stimulated by LSSmOrange, the other fluorescent protein. Of course, we checked also the emission and excitation of our BL21(DE3) cells (data not shown) and we can eliminate the theories that these cells disturb this florescence measurement.

Figure 2. Excitation spectrum (dashed line) and emission spectrum (solid line) of mKate with marked maximums

Figure 3. Overlay of excitation spectrum (dashed line) and emission spectrum (solid line) of mKate and LSSmOrange

Moreover, unlike for our LssmOrange BioBrick (BBa_K1055001), there is a crystal structure available for further analysis in the http://www.rcsb.org/pdb/explore.do?structureId=1UIS RCSB database which is shown below:

Crystal structure of mKate dimer with the RCSB code 1UIS.

The expression of mKate is not toxic towards the bacterial host E.coli Bl21(DE3). The growth rate was in a usual time scale of approximately 30 minutes.

Figure 4. Semi logarithmic scaled growth curves of E.coli BL21(DE3) WT, BL21DE3 mKate and BL21DE3 LSSmOrange. The ln (OD600 values) are plotted against the time [min].

References

Daria M. Shcherbakova et al. (2012) An Orange Fluorescent Protein with a Large Stokes Shift for Single-Excitation Multicolor FCCS and FRET Imaging. J. Am. Chem. Soc. 134 (18), 7913–7923

http://www.evrogen.com/products/basicFPs.shtml

Sequence and Features