Difference between revisions of "Part:BBa K1055001"
(6 intermediate revisions by the same user not shown) | |||
Line 2: | Line 2: | ||
<partinfo>BBa_K1055001 short</partinfo> | <partinfo>BBa_K1055001 short</partinfo> | ||
− | LssmOrange is a fluorophor derived from dsRed | + | LssmOrange is a fluorophor derived from dsRed and is a FRET partner of mKate [[https://parts.igem.org/Part:BBa_K1055000 BBa_K1055000]] |
− | + | ATTENTION: If you want to make a fusion protein with this part, please refer to our [https://parts.igem.org/Part:BBa_K1055001:Experience '''experiences''']! | |
===Usage and Biology=== | ===Usage and Biology=== | ||
+ | |||
+ | [[Image:LSSmOrange_bac.png|400px|right|thumb|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 [https://parts.igem.org/Part:BBa_K1055000 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. | ||
+ | [[Image:LSSmOrange_Diagramm.png|center|400px|thumb|Figure 2. '''Excitation spectrum (dashed line) and emission spectrum (solid line) of LSSmOrange with marked maximums''']] | ||
+ | [[Image:FRET_Normalized_2.png|center|400px|thumb|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: | ||
+ | |||
+ | [[Image:LssmOrange_first_activeHelix.png|center|400px|thumb|]] | ||
+ | |||
+ | 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. | ||
+ | |||
+ | |||
+ | [[Image:Grow_log.png|center|400px|thumb|center|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 | ||
+ | |||
+ | <!-- Add more about the biology of this part here | ||
+ | |||
<!-- --> | <!-- --> |
Latest revision as of 18:11, 12 October 2013
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
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.
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 570
- 1000COMPATIBLE WITH RFC[1000]