Difference between revisions of "Part:BBa K1055001:Experience"

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Applications of BBa_K1055001

When tried to fuse to the Tar receptor of E. coli C-terminally we encountered major clonation problems since our methods were not able to acces the DNA. After numerous trials and several methods, we performed an "m-fold" analysis of our hypothetical DNA construct. Please take a look below:

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

As shown in Fig. 1 and 4, 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

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