Part:BBa_K1055001

LssmOrange is a fluorophor derived from dsRed

LssmOrange is a fluorophor derived from dsRed and is a FRET partner of mKate [BBa_K1055000]

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

Usage and Biology

Figure 1. Pellet of E. coli BL21 (DE3) cells with expressed LSSmOrange. Left side without UV radiation, right side with UV radiation

LSSmOrange is an orange fluorescent protein that we want to use for FRET, we us it as a donor. LSSmOrange has an excitation maximum by 437 nm and an emission maximum by 572 nm. The excitation maximum is at 415 nm, another at 453 nm and our measured emission maximum is at 564 nm (Fig. 2). This aberration changes nothing on the FRET system. The excitation maximum is big enough to stimulate mKate, the other fluorescent protein (Fig. 3). Of course, we also checked the emission and excitation of our E. coli 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 LSSmOrange with marked maximums

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

Furthermore we did homology modeling with this part, please refer to our http://2013.igem.org/Team:TU_Darmstadt/modelling/Structure wiki page, whose best model results are illustrated below:

The expression of LSSmOrange 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 log scaled graph of E. coli BL21(DE3) growth rate expressing either LSSmOrange, mKate or being the wildtype.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 570
  
• 1000
  COMPATIBLE WITH RFC[1000]